Method of Providing Disease-Specific Binding Molecules and Targets

ABSTRACT

Provided are novel specific binding molecules, particularly human antibodies as well as fragments, derivatives and variants thereof that recognize neoepitopes of disease-associated proteins which derive from native endogenous proteins but are prevalent in the body of a patient in a variant form and/or out of their normal physiological context. In addition, pharmaceutical compositions comprising such binding molecules, antibodies and mimics thereof and methods of screening for novel binding molecules, which may or may not be antibodies as well as targets in the treatment of neurological disorders such as Alzheimer&#39;s disease are described.

RELATED APPLICATIONS

U.S. patent application Ser. No. 12/522,031, which is the National Stageof International Application No. PCT/EP2008/000053, filed Jan. 7, 2008,U.S. Provisional Application No. 60/934,291, filed Jun. 11, 2007, U.S.Provisional Application No. 60/878,831, filed Jan. 5, 2007, EuropeanApplication No. 07020341.9, filed Oct. 17, 2007, and EuropeanApplication No. 07000211.8, filed Jan. 5, 2007 are incorporated hereinby reference in their entireties.

The content of the electronically submitted sequence listing (Name:21591900006_sequencelisting_ascii.txt, Size: 46,754 bytes; and Date ofCreation: Mar. 13, 2013) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel specific binding molecules,particularly human antibodies as well as fragments, derivatives andvariants thereof that recognize disease-associated epitopes, includingneoepitopes, of proteins which derive from native endogenous proteins,and which are prevalent in the body of a patient in a variant formand/or out of their normal physiological context. In addition, thepresent invention relates to pharmaceutical compositions comprising suchbinding molecules, antibodies and mimics thereof, and to methods ofscreening for novel binding molecules, which may or may not beantibodies, targets and drugs in the treatment of various disorders, inparticular neurological disorders such as Alzheimer's disease,amyloidoses and beta-amyloid pathology.

BACKGROUND OF THE INVENTION

The success in generating monoclonal antibodies rests on the efficientand selective fusion of antigen-stimulated B cells with a murine myelomacell line followed by selection of stable antibody producing hybrids asoriginally described by Köhler and Milstein, Nature 256 (1975), 495-497.However, the therapeutic utility of murine based antibodies in human ishampered by the human anti-mouse antibody (HAMA) response in view oftheir non-human origin. Approaches for making human or human-likemonoclonal antibodies became available through genetic engineering.However, the methods hitherto available suffer from the drawback thatthey are not suitable to produce antibodies with the characteristics ofthose produced in the course of a physiological human immune response.Furthermore, such antibodies may not be specific enough because ofcross-reactivity with other proteins and/or the target protein incontext with normal physiological function. In case of Alzheimer's orParkinson's disease, for example, antibodies that also cross-react withhigh affinity with physiological derivatives of amyloid precursorprotein (APP) or alpha synuclein are considered to exhibit side effectsrelated to the normal functions of the physiologic target structures. Inthis respect, an undesired autoimmune disease would downrightly beinduced—a hardly calculable risk in the conceptual design of activeimmunization experiments employing protein structures that, in variantform, also occur physiologically. Side effects not related to the targetstructure are, for example, anaphylactic reactions, as are to beexpected as undesired and dreaded side effects of the systemicadministration of exogenous proteins. According to recent findings, thiscan also be the case in so-called humanized antibodies, which originallystem from non-human organisms, usually from mice. On the other hand,active immunization with pathological relevant antigens bears theconsiderable risk of patients developing antibodies and T cell responseswhich also recognize physiological variants of such proteins and inconsequence lead to a dangerous and uncontrollable autoimmune response.

Thus, there is a need of providing agents which are specific for atarget involved in a disorder and which are tolerated by the human body.

SUMMARY OF THE INVENTION

An object of the present invention is a method for identifying,validating and producing diagnostically and therapeutically usefulbinding molecules, in particular antibodies that are directed againstpathologic variants of endogenous proteins. More specifically, thepresent invention relates to a method of isolating a disease-associatedprotein-specific binding molecule comprising:

(a) subjecting a sample obtained from a patient who is symptom-free, orwho is clinically unusually stable, but who is affected with or at riskof developing a disorder to a specimen of pathologically altered cellsor tissues with predetermined pathological characteristics; and

(b) identifying and optionally isolating a binding molecule which bindsto said specimen but not to corresponding cells or tissues without suchpathological characteristics as it may be derived from a healthysubject.

Known is the fact that, in case of autoimmune diseases, antibodies aredirected against autologous cells and proteins or other compounds suchas glycolipids expressed by said cells while evading the known tolerancemechanisms. Also known is the fact that, in case of endogenousneoplastic developments, a cellular and humoral immunity to theneoplastic cells can develop and can thus effect an endogenousimmunological protection mechanism against neoplastic tissuedegeneration.

The present invention makes use of the surprising finding thatantibodies can also be directed against pathophysiologically relevantvariants of endogenous proteins, in particular against neoepitopes,which are formed due to pathologically altered transcription,translation, or post-transcriptional or post-translational modification,or proteolytic processing, or aggregation. Such antibodies are directedagainst endogenous proteins which, owing to their new structure thatdeviates from the normal physiology, become pathophysiologicallyrelevant by means of developing pathological effects. For reasons ofimmune tolerance, the antibodies connected with the corresponding immuneresponse to neoepitopes in such pathological variants do not normallyexhibit any cross reactions against the physiologically functionalproteins, however, as opposed to the case of autoimmune diseases. Thisis because the formation of potentially cross-reactive antibodies isspecifically suppressed by the known tolerance mechanisms, whereas thedevelopment of an immune response to pathological neoepitopes can escapetolerance.

Hence, the present invention relates to a novel approach of identifyingdiagnostically, therapeutically, and preventively active bindingmolecules, especially antibodies and antibody fragments from clinicallypreselected human subjects by means of interaction with identifiablepathological structures.

The present invention is thus directed to antibodies or antigen-bindingfragments and similar antigen binding molecules which are capable ofrecognizing epitopes, including neoepitopes, of disease-associatedproteins which derive from native endogenous proteins and are prevalentin the body of a patient in a variant form, e.g. as a pathologicalprotein and/or out of their normal physiological context. Furthermore,the present invention relates to compositions comprising said antibodiesand to immunotherapeutic and immunodiagnostic methods using the same.

Furthermore, in antibody identification, the method according to thepresent invention can do without previous hypothesis on the identity ofits molecular target structure, solely by means of its association withpathologically relevant structures. Besides the possibility of thusidentifying molecular target structures hitherto unknown for specificdiseases, a further advantage of antibodies that are exclusivelydirected against pathological structures is based on the fact that theirpharmacodynamic availability is not negatively influenced by binding tonon-diseased tissues in such a way that the antibody is buffered withrespect to its concentration and sink effects thus hampering thedetermination of therapeutically effective concentrations. Furthermore,the antibody and binding molecules of the present invention arepreferably characterized in that they react with the variant form of thedisease-associated protein in viva or with a cell or cell membrane, andon a section of the pathologically characterized diseased tissue,respectively, but not or to a significantly lesser extent with thephysiological variant of the cognate protein; see also, e.g., Example 2.

Since the present invention enables identifying and isolating moleculartarget structures in diseased cells and tissues, a further embodimentconcerns the antigen and pathological protein, i.e. disease-associatedprotein, respectively, which is bound by the neoepitope-specificantibody of the present invention.

A particularly preferred embodiment is a human antibody orantigen-binding fragment thereof which demonstrates the immunologicalbinding characteristics of any of the antibody characterized by thevariable regions VH and/or VL as set forth in Tables 2 and 3, infra.Alternatively, the antibody is a humanized, xenogeneic, or a chimerichuman-murine antibody, the latter being particularly useful fordiagnostic methods and studies in animals. Therapeutic compositionsincluding the antibody or active fragments thereof, or agonists andcognate molecules, or alternately, antagonists of the same, and methodsof use of such compositions in the prevention, diagnosis or treatment ofa disease using these compositions are also included, wherein aneffective amount of the composition is administered to a patient in needof such treatment.

The antigen-binding fragment of the antibody can be a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F(ab′)₂fragment, or any other antigen-binding fragment. In a specificembodiment, infra, the antibody or fragment thereof is a human IgGisotype antibody.

Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that produces the antibodyhaving the distinct and unique characteristics as defined below.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Preferably, said variable region comprises at least onecomplementarity determining region (CDR) of the VH and/or VL of thevariable region as set forth in Tables 2 and 3, infra. A correspondingset of CDRs is given in Table 4, infra.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for neoepitopes that are indicative and/orcausative for a disorder, in particular for a disorder of the brain suchas Alzheimer's disease and Parkinson disease.

The antibody, immunoglobulin chain(s), binding fragments thereof andantigen binding to said antibody can be used in pharmaceutical anddiagnostic compositions for immunotherapy and diagnosis, respectively.The use of the foregoing compositions in the preparation of a medicamentis however preferred.

Hence, it is a particular object of the present invention to providemethods for treating or preventing a neurological disorder characterizedby abnormal accumulation and/or deposition of a protein in the centralnervous system without interfering with the natural function of therespective protein. The methods comprise administering an effectiveconcentration of an antibody or antibody derivative to the subject wherethe antibody binds to the pathological form of the protein or theprotein deposit with a substantially higher affinity than to the normalphysiological form of the protein. In a preferred embodiment, thepresent invention provides methods for treating or preventing or slowingthe onset of diseases associated with the accumulation and deposition ofthe amyloid beta peptide in a subject, such as Alzheimer's disease,Down's syndrome, mild cognitive impairment, cerebral amyloid angiopathy,vascular dementia, multi-infarct dementia. The methods compriseadministering an effective concentration of an antibody or antibodyderivative to the subject where the antibody binds to the pathologicalform of the protein or the protein deposit with higher affinity than tothe normal physiological form of the protein. Similar therapeuticapproaches are envisaged for the treatment of Parkinson's disease,Huntington's disease, Creutzfeldt-Jakob disease, cystic fibrosis,Gaucher's disease and the like.

Further embodiments of the present invention will be apparent from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Antibody against beta-amyloid. A: Human antibodies. B: Controlstaining with known antibody against human beta-amyloid. Clinicallyunusually stable patients with Alzheimer's disease contain antibodies tobeta-amyloid plaques. Immunohistochemical staining with antibodies fromclinically unusually stable patients on brain sections obtained frompatients with pathologically confirmed Alzheimer's disease revealsantibodies that bind to beta-amyloid plaques confirmed by a knownantibody against human beta-amyloid.

FIG. 2: Antibody against neurofibrillary tangles. A: Human antibodies.B: Control staining with known antibody against human tau. Healthy humansubjects contain antibodies to neurofibrillary tangles.Immunohistochemical staining with antibodies from healthy subjects onbrain sections obtained from patients with pathologically confirmedAlzheimer's disease reveals antibodies that bind to neurofibrillarytangles confirmed by a known antibody against human tau.

FIG. 3: Antibody against dystrophic neurites. A: Human antibodies. B:Control staining with known antibody against human tau. Healthy humansubjects contain antibodies to dystrophic neurites. Immunohistochemicalstaining with antibodies from healthy subjects on brain sectionsobtained from patients with pathologically confirmed Alzheimer's diseasereveals antibodies that bind to dystrophic neurites.

FIG. 4: Antibody against beta-amyloid. The figure shows specific bindingof recombinant human NI-101.11 antibody that was isolated from aclinically unusually stable Alzheimer's disease patient to brainbeta-amyloid plaques. Brain sections obtained from a patient withneuropathologically confirmed Alzheimer's disease were stained withrecombinant human antibody at the indicated concentrations. Antibodybinding to beta-amyloid plaques with concentrations of 50 pM suggesthigh affinity binding.

FIG. 5: Binding of recombinant human NI-101.11 antibody to beta-amyloidplaques is not competed by linear synthetic N-terminal Abetapolypeptides. Binding of the recombinant antibody against brainbeta-amyloid (0.5 nM) cannot be competed by N-terminal Abeta-derivedpolypeptide representing positions 1 to 16 at concentrations up to 1 μM.

FIG. 6: Recombinant human NI-101.11 antibody recognizes a conformationalAbeta epitope that is not present in monomeric Abeta. Binding ofNI-101.11 to beta-amyloid plaques on brain sections can be competed byAbeta1-42 fibrils but not linear synthetic Abeta1-42 monomers.

FIG. 7: Recombinant human NI-101.11 antibody does not bind to linear,monomeric synthetic Abeta on Western blots. Preparations of monomericAbeta were separated by non-denaturing PAGE. Blotted protein was probedwith human recombinant antibody against beta-amyloid and controlantibodies against N-terminal linear Abeta sequences (6E10). No bindingof NI-101.11 to monomeric Abeta was detected. This observation suggeststhat the antibody recognizes a conformational Abeta epitope.

FIG. 8: Human NI-101.11 antibody binds artificial amyloid fibrilsprepared from synthetic Abeta1-42 peptides. Synthetic Abeta fibrils ormonomeric synthetic Abeta coated onto ELISA plates at equal coatingdensities were incubated with recombinant human antibodies against brainbeta-amyloid at the indicated concentrations. Binding activity of humanantibody against brain beta-amyloid to artificial amyloid fibrils (opensquares) is more than 100 times higher as compared to monomeric Abeta(filled squares). Control antibody 22C4 preferentially binds tomonomeric Abeta (filled cicles), and less well to fibrils (opencircles). This suggests that NI-101.11 recognizes a conformationalepitope which is also present on artificial amyloid fibrils preparedfrom synthetic Abeta peptides.

FIG. 9: Absent cross-reactivity of recombinant human NI-101.11 antibodyto cellular full-length APP or with any of its physiological derivativesoccurring in cultured cells. In contrast to the control antibody (6E10)that binds to cell-surface APP, binding of NI-101.11 to full-length APPpresent at cellular surfaces is absent. These data demonstrate absentcross-reactivity of NI-101.11 to physiological, cellular full-lengthAPP.

FIG. 10A-C: Absence of binding of NI-101.11 to monomeric Abeta via sizeexclusion chromatography. FIGS. 10A and 10B show no binding of NI-101.11or an unrelated control antibody to monomeric FITC-labeled Abeta1-42while FIG. 10C shows prominent binding of antibody 22C4 that recognizesa linear epitope present in the C-terminus of Abeta.

FIG. 11: Competition ELISA showing that binding of antibody 6E10, anantibody directed against a linear epitope at the N-terminus of Abetacould be completely blocked upon pre-incubation with excessconcentrations of monomeric Abeta peptides while pre-incubation withexcess concentrations of these monomeric Abeta peptide preparations didnot abolish NI-101.11 binding.

FIG. 12: Binding of NI-101.13A and NI-101.13B to brain sections obtainedfrom Tg2676 transgenic mouse model of Alzheimer's disease.

FIG. 13: ELISA showing preferential binding of NI-101.13A and NI-101.13Bto artificial amyloid fibrils as compared to monomeric Abeta.

FIGS. 14A-B: FIG. 14A shows the binding of recombinant NI-101.12 tosynthetic Abeta1-42 peptide via ELISA. FIG. 14B shows NI-101.12 bindingwas competed by excess Abeta1-42 peptide.

FIG. 15: Recombinant human NI-101.11 antibody against brain beta-amyloidcrosses the blood brain barrier in a transgenic mouse model ofAlzheimer's disease, and binds to brain beta-amyloid plaques in vivo.

FIGS. 16A-B: Recombinant human NI-101.11 antibody improves abnormalcognitive behavior in a transgenic mouse model of Alzheimer's disease.24 months old arc Abeta mice were treated weekly i.p. with 3 mg/kgantibody for 2 months. Y-maze behavioral testing was performed beforeand after completion of the treatment.

FIG. 17: Blood-brain barrier penetration and decoration of amyloidplaques by peripherally administered NI-101.11. NI-101.11 can cross theblood-brain barrier and bind to beta-amyloid deposits in NI-101.11treated mice (left panel) whereas no such staining is visible in animalstreated with the human control antibody (right panel). Recombinant humanNI-101.11 antibody reduces brain beta-amyloid plaque load after systemictreatment for two months.

FIG. 18: Passive immunization with NI-101.11 reduces beta-amyloid loadin arc Abeta mice. (A, B) Thioflavin S and Congo Red plaque loadanalyses reveal significant reductions of more than 50% compared to thecontrol antibody treated animals (Mann-Whitney U; p=0.02 for cortex,p=0.009 for hippocampus for ThioS and p=0.009 for cortex and p=0.04 forhippocampus for Congo Red analysis). Scale bar: 200 μm. (C-E) ThioflavinS analysis reveals a significant reduction in beta-amyloid burden (C),number of beta-amyloid plaques (D) and average plaque size (E) inNI-101.11 treated arc Abeta mice compared to control treated animals.Mann-Whitney U statistics: p=0.02 for plaque area cortex; p=0.009 forplaque area hippocampus; p=0.047 for plaque number cortex; p=0.047 forplaque number hippocampus; p=0.009 for plaque size cortex; p=0.009 forplaque number hippocampus.

FIG. 19: Reduced beta-amyloid load is accompanied by decreasedastrocytosis and microgliosis A) Quantification of anti-GFAP stainingrevealed a significant reduction in the number of reactive astrocytes inthe cortex of NI-101.11 treated arc Abeta mice when compared to controltreated transgenics. B) Quantification of Iba-1 staining showed a trendtowards a reduced number of activated microglia in NI-101.11 treatedmice in cortex and hippocampus. Scale bar: 200 μm.

FIG. 20: No increase of brain microhemorrhages after two months oftreatment with recombinant human NI-101.11 antibody. 24 months old arcAbeta mice with proven massive congophilic amyloid angiopathy weretreated weekly i.p. with 3 mg/kg antibody for 2 months. Representativepicture of a brain microhemorrhage in arc Abeta mice revealed by Perl'sPrussian blue staining (left). Quantitative analysis demonstrates asignificantly elevated frequency of micorhemorrhages in arc Abetatransgenic mice compared to their wildtype littermates. Chronictreatment with NI-101.11 did not result in increased frequency ofmicorhemorrhages. Scale bar: 20 μm

FIG. 21: Recombinant human NI-101.11 antibody inhibits the formation ofsynthetic Abeta fibrils in vitro. The effect of recombinant humanNI-101.11 antibody on the formation of Abeta fibrils was assayed bymeasuring Thioflavin S bound to aggregated Abeta by fluorescenceanalysis.

FIG. 22: Antibody-mediated dose-dependent phagocytosis of FITC-Abeta1-42fibrils by BV-2 microglial cells was measured upon inhibition of thescavenger receptor system. NI-101.11 triggers potent dose-dependentFcgamma receptor-mediated phagocytosis of Abeta fibrils.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or antibody polypeptides of thepresent invention include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native bindingmolecule, antibody, or polypeptide. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of antibodies and antibody polypeptides ofthe present invention include fragments as described above, and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants may occur naturally orbe non-naturally occurring Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of neoepitope-specific bindingmolecules, e.g., antibodies and antibody polypeptides of the presentinvention, are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of abinding molecule or fragment thereof, an antibody, or an antibodypolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those peptides which contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refer to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein. A “binding molecule” as used in the context ofthe present invention relates primarily to antibodies, and fragmentsthereof, but may also refer to other non-antibody molecules that bind toa neoepitope including but not limited to hormones, receptors, ligands,major histocompatibility complex (MHC) molecules, chaperones such asheat shock proteins (HSPS) as well as cell-cell adhesion molecules suchas members of the cadherin, intergrin, C-type lectin and immunoglobulin(Ig) superfamilies. Thus, for the sake of clarity only and withoutrestricting the scope of the present invention most of the followingembodiments are discussed with respect to antibodies and antibody-likemolecules which represent the preferred binding molecules for thedevelopment of therapeutic and diagnostic agents.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is an antigen-binding moleculewhich comprises at least the variable domain of a heavy chain, andnormally comprises at least the variable domains of a heavy chain and alight chain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Thisquaternary antibody structure forms the antigen binding site present atthe end of each arm of the Y. More specifically, the antigen bindingsite is defined by three CDRs on each of the VH and VL chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to an antigen is denoted herein interchangeably asan “antigen binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table I as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are antigen-binding fragments also comprisingany combination of variable region(s) with a hinge region, CH1, CH2, andCH3 domains. Antibodies or immunospecific fragments thereof of thepresent invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human patients, human immunoglobulin librariesor from animals transgenic for one or more human immunoglobulins andthat do not express endogenous immunoglobulins, as described infra and,for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. A humanantibody is still “human” even if amino acid substitutions are made inthe antibody, e.g., to improve binding characteristics.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a VL or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofAβ.

The term “neoepitope” in accordance with the present invention denotesan epitope which is unique for a disease pattern and contained in orformed by a disorder-associated protein which is a pathological variantfrom an otherwise non-pathological protein and/or deviating from thephysiology of the healthy state. Said pathophysiological variants can beformed by means of pathologically altered transcription, pathologicallyaltered translation, post-translational modification, pathologicallyaltered proteolytic processing, pathologically altered complex formationwith physiological or pathophysiological interaction partners orcellular structures in the sense of an altered co-localization, orpathologically altered structural conformation—like for exampleaggregation, oligomerization or fibrillation—whose three- orfour-dimensional structure differs from the structure of thephysiologically active molecule. Moreover, a pathophysiological variantcan also be characterized in that it is not located in its usualphysiological environment or subcellular compartment. As an example,neoepitopes may be located in the pathologically conspicuous structuresin the areas of brain tissues that obviously experience or have alreadyexperienced functional damage. Whether a given structure, for examplecell or tissue, or protein displays a neoepitope can be verified byreversing the method described below for isolating and characterizing adisorder-associated protein specific binding molecule in that a bindingmolecule, for example antibody identified by said method is used toscreen a sample for binding to the antibody, thereby determining thepresence of a neoepitope.

The phrases “disease-associated protein specific” and “neoepitopespecific” are used interchangeably herein with the term “specificallyrecognizing a neoepitope”. As used herein terms such as “absence ofcross-reactivity”, “specific,” “specifically recognizing,” “specificallybinding,” “preferentially binding,” and the like refer to the bindingmolecule's ability to discriminate between the neoepitope of adisorder-associated protein and the native protein in its wild type formand natural context. Thus, the binding molecule of the present inventionhas a preferential binding affinity to the neoepitope over the nativeprotein antigen by a factor of at least two, preferably at least 5,usually more than by a factor of 10, particularly preferred by a factorof 50 and even more preferred higher than 100. Furthermore, the relativeKD of the binding molecule, e.g., antibody for the specific targetepitope, e.g. neoepitope is preferably at least 10-fold less, morepreferably at least 100-fold less or more than the KD for binding thatantibody to other ligands or to the native counterpart of thedisease-associated protein.

By “specifically binds,” or “specifically recognizes,” usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen binding domain,and that the binding entails some complementarity between the antigenbinding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

By “preferentially binds,” it is meant that the binding molecule, e.g.,antibody specifically binds to an epitope more readily than it wouldbind to a related, similar, homologous, or analogous epitope. Thus, anantibody which “preferentially binds” to a given epitope would morelikely bind to that epitope than to a related epitope, even though suchan antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind a targetpolypeptide disclosed herein or a fragment or variant thereof with anoff rate (k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹,5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. More preferably, an antibody of theinvention may be said to bind a target polypeptide disclosed herein or afragment or variant thereof with an off rate (k(off)) less than or equalto 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹,10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or  ⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind a targetpolypeptide disclosed herein or a fragment or variant thereof with an onrate (k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 510³ M⁻¹ sec⁻¹,10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of theinvention may be said to bind a target polypeptide disclosed herein or afragment or variant thereof with an on rate (k(on)) greater than orequal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen. See, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit. The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein the term “properly folded polypeptide” includespolypeptides in which all of the functional domains comprising thepolypeptide are distinctly active. As used herein, the term “improperlyfolded polypeptide” includes polypeptides in which at least one of thefunctional domains of the polypeptide is not active. In one embodiment,a properly folded polypeptide comprises polypeptide chains linked by atleast one disulfide bond and, conversely, an improperly foldedpolypeptide comprises polypeptide chains not linked by at least onedisulfide bond.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two ore more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which themanifestation of the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

II. Methods to Identify Binding Molecules

The present invention generally relates to means and methods fordiscovering therapeutically efficient antibodies from clinicallypreselected human subjects. As demonstrated in the examples, humanantibodies against abnormal structures in human brain diseases can beisolated from phenotypically healthy, or clinically unusually stablepatients and corresponding recombinant antibodies can be successfullyused for the treatment, amelioration of pathology and prevention ofimpairment of brain function without substantial side effects. Clinicalstability or non-progression of disease can be identified by way ofexample by measurement over time of clinical, e.g., cognitive, status(by neuropsychological testing, for example); assessment of the globalfunctional level; evaluation of the daily living capacities orbehavioral deficits; volumetric analysis of brain structures; in vivomeasurement of pathological deposits of abnormal proteins in brain (e.g.PET beta-amyloid imaging) or biochemical variables in body fluids (e.g.tau proteins, or Abeta peptides); and by comparison to the naturalcourse/history of the disease. Thus, the present invention providesantibodies and binding molecules which are capable of recognizingneoepitopes of disease-associated proteins which derive from nativeendogenous proteins, and are prevalent in the body of a patient in avariant form, e.g. as a pathological protein and/or out of their normalphysiological context. In particular, antibodies and antigen-bindingfragments thereof are provided, which demonstrate the immunologicalbinding characteristics and/or biological properties as outlined for theantibody illustrated in the Examples. Where present, the term“immunological binding characteristics,” or other bindingcharacteristics of an antibody with an antigen, in all of itsgrammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.Naturally, the present invention extends to the antibody producing celllines and recombinant cells as well. The present invention furtherrelates to diagnostic assays and kits that comprise the binding moleculeof the present invention and to therapeutic methods and therapeuticevaluations based thereon.

The present invention is based on the observation that in human subjectsand patients preselected according to specific clinical criteria, whobear a risk of developing a neurological disorder like Alzheimer'sdisease due to their age, as related to the humoral defense, antibodiesto endogenous pathophysiological variants like Abeta peptide aggregates,neurofibrillary tangles, dystrophic neurites and further cellularstructures can also be found, which are neuropathologicallycharacteristic for the disorder. These structures can be found alone orin combination with other pathological structures and can, as in case ofthe Abeta aggregates and the precursors of the neurofibrillary tangles,develop pathological effects. However, those antibodies specificallyrecognizing said structures exhibit no or significantly lowercross-reactivity to the normal physiologically functional forms of theproteins underlying the pathological structures, contrary to the knowncase of an autoimmune response.

Without intending to be bound by theory it is, based on the experimentsperformed in accordance with the present invention, believed that adisorder does not have to become manifest and phenotypicallyperceivable, as in case of an infection, in order to result in anexperimentally measurable activity of the humoral immune system like thegeneration or activation of specific B cells or B memory cells. In caseof tumor cells or differentiated cells, which have to be physiologicallydisposed of, mechanisms are already known, wherein partners like T4helper cells and cytotoxic T cells and natural killer cells of thecellular immune system cooperate in the induction of apoptosis. It hasto be assumed that, also in a healthy human, tumor cells or precursorsthereof are formed on a daily basis by means of mutation. However, theseare immediately driven to apoptosis by means of humoral and cellularmechanisms, so that a tumor is not detectable. In this sense, a“healthy, or clinically unusually stable, patient” is not understood todenote an individual in whom no disease events take place, but anindividual in whom the diverse disease events are controlled by blockingor defense mechanisms before they phenotypically manifest themselves.

In view of the above, it was hypothesized in accordance with the presentinvention that it should be possible to identify an antibody or anantibody-producing cell from specific patient collectives or healthysubjects, which have been preselected according to clinical criteria,without precognition of the molecular nature of a target structureepitope, e.g. neoepitope, wherein the antibody, in case of alreadyprevailing tolerance in the donor organism—which is, for example, provedby the absence of autoimmunity—could successfully be employed against adisease in form of a recombinantly produced agent. Identifying such anantibody can thus be conducted without precognition of the molecularepitope of the target structure, but rather solely by means of bindingto neoepitopes of pathologically conspicuous structures inclinico-pathologically well characterized tissue sections derived fromhuman patients or from animal models of the respective disease.

Accordingly, in a first aspect the present invention relates to a methodof isolating a disorder-associated protein-specific binding moleculecomprising:

(a) subjecting a sample obtained from a patient who is symptom-free, orwho is clinically unusually stable, but who is affected with or at riskof developing a disorder or effectively suppressing the manifestation oroutbreak of a disorder to a specimen of pathologically orphysiologically altered cells or tissue of predetermined clinicalcharacteristics; and

(b) identifying and optionally isolating a binding molecule whichpreferentially binds to said specimen but not or with significantlylower affinity to corresponding cells or tissues without suchpathological characteristics as it may be derived from a healthysubject.

The method of the present invention can be performed as outlined in theExamples section with means well known to a person skilled in the art.For example, a liquid sample obtained from the patient can be passedthrough a first aperture of a duct which is in contact with the specimentarget structure firmly held in an object holder, thereby allowingputative binding molecules present in the sample, either in a solubleform or expressed on the cell surface and membrane, respectively, tobind to said target structure. The liquid sample may contain for examplelymphocytes and/or antibodies while the specimen may be a tissue sectionor a membrane coated with molecules or molecular combinations which aredistinct for a pathological target structure.

Any non-binding matter can be removed via the second duct aperture. Atthe same time, the temperature of the object holder may be controlled byan object holder thermostat, for example at a temperature at whichnatural binding of the putative binding molecule to the neoepitope ofthe antigen specific for the specimen takes place in the human body. Byway of the flowing motion, e.g. passing the liquid sample containingbinding molecules, preferably at body temperature over the targetstructure natural systems of binding interactions can be simulated.However, other methods of incubating the sample with the specimen suchas by means of a shaker or rotating table may be used as well. Aparticular advantage of the above-mentioned system is that it allows aninterruption of metabolic processes at any time by decreasing thetemperature of the object holder by means of the object holderthermostat. In doing so, the temperature of the object holder can bedecreased to for example 2-10° C., in particular 4° C. A correspondingdevice that can be used in accordance with the method of the presentinvention is described in European patent application EP 1 069 431 A2.Hence, the method of the present invention will allow identification andcharacterization of the binding partners as well as at the same time toidentify and characterize the molecular classes, molecular groups and/ormolecular parts required for the binding process, i.e. the targetstructures of the specimen, which hitherto may be unknown. This will notonly open up new possible ways of diagnosis, but will also provide a newtest system for therapeutic approaches on a molecular level.

As a patient may qualify in accordance with the present invention a poolof healthy volunteers if specific surrogate markers predict a highprobability of a status of a disease, which has surprisingly—andpossibly due to a specific endogenous immune response—not becomeclinically manifest, however. In this sense, as related to diversediseases, a healthy elderly human would be an individual, in which aneurodegenerative disease like Alzheimer's disease or Parkinson'sdisease is not yet clinically manifest, but in which the preclinicaldevelopment of pathophysiological protein variants, i.e.disorder-associated proteins, and in the above-mentioned sense by meansof early intervention of the humoral immune system with or withoutinvolvement of cellular components of the immune system, has beenrestricted or delayed to such an extent that, until the moment when thehealthy, but not preclinical patient participated in the study, theclinical manifestation of the disease had not yet occurred. Preferably,inconspicuous volunteers in whom neither autoimmunological processeshave been diagnosed nor other possible pathological conditions occur asside effects are recruited as donors for the sample in the firstinstance.

In principle samples from patients may be used, who have undergone anactive immunization with variants of physiological proteins or peptides,wherein the antibody development has been boosted by the immunization.Antibodies, for example, can be identified and isolated from Alzheimer'sdisease patients that have been vaccinated with Abeta peptide. See fore.g., Hock et al., Nature Medicine 8:1280-1275 (2002), Hock et al.,Neuron 38:547-554 (2003), and WO 2004/095031, each incorporated hereinby reference in their entireties. However, it may be preferred to usesamples from volunteers which have not received such immunization orcorresponding medication concerning the disorder the variantpathological protein is associated with and/or causative of.

According to the present invention, samples of a patient, e.g. ofindividuals that have been clinically preselected are analyzed for thepresence of binding molecules specifically recognizing specimen ofpathologically conspicuous structures, for example in ex vivo tissuefrom clinico-pathologically characterized human patients or animalmodels like, for example, transgenic mice, or in vitro cell structures,or in pathological allogenic or xenogenic tissue. Preferably, saidpatient and/or as said subject providing the specimen are human, mostpreferably both.

The characteristic pathologically or physiologically altered sample,cell or tissue specimen is preferably displayed by optical detectionafter reaction with a binding molecule, e.g. antibody of the presentinvention. The specimen may be obtained as/from a cell sample, tissuesection, cellular smear test, cell or tissue sample of an animal modelof a human disease or in vitro cultured cell and tissue material.Preferably, at least one of said specimen is present in an object holderin form of a scan positions, wherein each scan position corresponds to aspecific pathological structure and at least two of the scan positionsare concomitantly exposed to detection of a reaction with the antibody.By way of example, antibodies to the neuropathological hallmarks of suchneurodegenerative diseases as Alzheimer's disease, Parkinson's diseaseor tauopathies—including beta-amyloid plaques, Lewy bodies,neurofibrillary tangles and dystrophic neuritis can be detected onsections obtained from human post-mortem brain tissues withclinico-pathologically confirmed diagnoses of the respective diseaseentities. This is done by mounting small rods of paraffin embeddedtissue on to glass slides which are then probed with samples of apatient or healthy donor. The detection of a reaction with the antibodyis done following standard procedures of immunohistochemistry andmicroscopical scanning.

Multiple tissue microsections containing various human disease tissuescan be assembled on the glass slide to form a multiple tissuemicroarray. Similarly, additional specimens from tissue samples ofanimal models of human disease, cellular smear or cells can be embeddedin paraffin and mounted on glass slides alone or in combination withabove described human post-mortem Alzheimer's disease brain tissue ortissue arrays. Thus, a single test position on the glass slide cancomprise mixed arrays of tissue and other specimen displayingpathologically conspicuous structures.

In order to increase the throughput of the assay, more than one testposition is mounted on a glass slide. Preferably, eight test positionsare mounted onto glass slides in a two by four format fitting the 96well or microtiter format. A more detailed description of thismicrotiter-compatible tissue microarray can be found infra in thesupplementary methods section below.

As mentioned the sample to be analyzed may comprise a body fluid, a cellsample or the supernatant of a cell sample or a derivative thereof. Bodyfluids such as lumbar cerebrospinal fluid (CSF), plasma or urine can becollected following standard clinical procedures after informed consentof the patients. Most preferably, the sample comprises or is derivedfrom B-cells or memory B-cells and/or comprises antibodies.

Preferably, said patient has been determined to be affected with a notyet manifested disorder or at risk to develop the disorder by thepresence or absence of a surrogate marker, or by an unusually stabledisease course. Clinical criteria are to be considered in connectionwith surrogate markers having either an increased probability of theoccurrence or the manifestation of a disease according to the presentinvention in the sense of a preclinical condition, or, vice versa,proving the improbability of such a disease already having occurred as,for example, a genetic constellation promoting the disease is existentor an extreme exposition or way of living renders the phenotypicaldevelopment of a disease probable. In the case of Alzheimer's diseasethis means that, according to the present invention, such humanvolunteers are searched for B cells or memory B cells againstneuropathology-associated protein complexes like Abeta aggregates,oligomeric Abeta species and beta-amyloid fibrils in beta-amyloidplaques, for tau filaments in neurofibrillary tangles, for alphasynuclein in Lewy bodies or components hitherto not molecularlyidentified that, with respect to age, belong to a group of individualsin whom either the prevalence of Alzheimer's disease is particularlyhigh or who originate genetically from a population bearing a high riskfor Alzheimer's disease. These are, for example, persons older than 75years having no or only marginal neuropsychologically measurablecognitive impairments, or in case of tumor indications, persons havinghighly indicative tumor markers, for example genetic tumor markers, butnot suffering from the disease (Alloul et al., Arch. GerontologyGeriatrics 27 (1998), 189; Dunn et al., Immunity 21 (2004) 137-148). Incase of mild cognitive impairment, these are patients that have remainedclinically, neuropsychologically, or cognitively stable for years inspite of their annual statistical risk of more than 20% for developing aneurodegenerative disease that is clinically manifest and progressive inthe further course. Thus, in one embodiment said surrogate marker isselected from the group consisting of old age, brain amyloid load, ApoEgenotype, APP genotype, PSI genotype, levels in body fluids of Abetapeptide, isoprostanes, Tau, and phospho-Tau.

A particular approach in employing the method according to the presentinvention is testing samples of B cells and B memory cells fromclinically preselected volunteers against arrays of specimen ofpathologically conspicuous tissues of entirely different or relatedprimary diseases. In particular, such pathological tissues originatefrom human patients suffering from neurodegenerative diseases,protein-misfolding diseases, tissue amyloidoses and other diseasesrelated to pathological deposits, autoimmune disorders, inflammatorydiseases, hyperproliferative and neoplastic diseases, e.g., tumors,storage diseases and inclusion diseases as well as from animal models ofhuman diseases, in particular from mice altered with pathologicallyrelevant human genes.

In one particularly preferred embodiment, the present invention focuseson Alzheimer's disease. In this embodiment, the sample is obtained froma subject preferably fulfilling the following criteria:

-   a) being 65, preferably 70 and more preferably 75 years of age or    older;-   b) having full cognitive capacity and good health; and-   c) having no clinical signs of dementia; or-   d) having unusually slow rates of progression of disease despite the    presence of an established clinical diagnosis of probable    Alzheimer's disease; or-   e) having unusually low conversion rates from Mild Cognitive    Impairment (MCI) to full blown Alzheimer's disease.

In a further embodiment, the method of the present invention furthercomprises the steps of:

-   a) purifying B cells or B memory cells from a sample which has been    identified to contain binding molecules, e.g. antibodies which    preferentially bind to said specimen but not or with significantly    lower affinity to corresponding cells or tissue of a healthy    subject;-   b) obtaining the immunoglobulin gene repertoire encoding said    antibodies from said B cells or B memory cells; and-   c) using said repertoire to express said antibodies, and optionally    wherein step (b) comprises the steps of:-   d) obtaining mRNA from said B cells or memory B cells;-   e) obtaining cDNA from the mRNA of step (iv); and-   f) using a primer extension reaction to amplify from said cDNA the    fragments corresponding to the heavy chains (HC) and the    kappa/lambda light chains (LC) of said antibodies.

Methods of producing clones of an immortalized human B cell and B memorylymphocyte, comprising the step of transforming human B memorylymphocytes using Epstein Barr Virus (EBV) in the presence of apolyclonal B cell activator are summarized in international applicationWO2004/076677. This international application also describes methods forobtaining a nucleic acid sequence that encodes an antibody of interest,comprising the steps of preparing an immortalized B cell clone andobtaining/sequencing nucleic acid from the B cell clone that encodes theantibody of interest and further inserting the nucleic acid into orusing the nucleic acid to prepare an expression host that can expressthe antibody of interest, culturing or sub-culturing the expression hostunder conditions where the antibody of interest is expressed and,optionally, purifying the antibody of interest. It goes without sayingthat the nucleic acid may be manipulated in between to introducerestriction sites, to change codon usage, and/or to add or optimizetranscription and/or translation regulatory sequences. For example,nucleic acid sequences can be generated by back-translation of thepolypeptide sequences of the present invention using software such asvector NTI software to generate nucleic acid sequences that arecodon-optimized and optimized for RNA stability. All this techniques arestate of the art and can be performed by the person skilled in the artwithout undue burden. Additional methods of immortalizing human B cellsare well known in the art, e.g., the construction of human hybridomas orhuman-murine chimeric hybridomas.

In a further aspect, the present invention relates to a binding moleculewhich is capable of selectively recognizing an epitope of adisease-associated protein including a neoepitope of adisease-associated protein, which preferably can be obtained orvalidated by the method of the present invention described hereinbeforeand illustrated in the examples. Advantageously, the binding molecule ofthe present invention does not substantially recognize said protein inits non-disorder-associated form; see also supra.

Means and methods for the recombinant production of binding molecules,in particular antibodies and mimics thereof as well as methods ofscreening for competing binding molecules, which may or may not beantibodies, are known in the art and are summarized, for example, ininternational application WO2006/103116 with respect to antibodiesagainst beta-amyloid and the treatment/diagnosis of Alzheimer's disease,the disclosure content of which is incorporated herein by reference forthis purpose of antibody engineering and administration for therapeuticor diagnostic applications.

However, as described herein, in particular with respect to therapeuticapplications in human the antibody of the present invention is a humanantibody. In this context, the variant pathological protein recognizedby the antibody is preferably associated with a neurological disorder,preferably a disorder of the brain.

Moreover, as demonstrated in Examples 3 to 5 the binding molecule of thepresent invention, in particular an antibody has several advantageousbiological properties one or more of which have been accomplished by thepresent invention for the first time, e.g. it is capable of:

-   (i) crossing the blood brain barrier, for example at the site of the    pathological event;-   (ii) binding beta-amyloid plaques, cerebrovascular amyloid, diffuse    Abeta deposits, neurofibrillary tangles, hyperphosphorylated tau,    alpha-synuclein positive Lewy-bodies or protein aggregates    associated with dystrophic neurites;-   (iii) removing beta-amyloid plaques in the brain and/or preventing    the formation of amyloid plaques in the brain;-   (iv) substantially restoring normal behavior; and/or-   (v) causing no microhemorrhages.

In a particular preferred embodiment, the antibody or equivalent bindingmolecule of the present invention may be distinguished from otherantibodies by one or more of the following properties, e.g. they areable to:

1. pass, at least in small amounts, the blood-brain barrier at the siteof the pathological events;2. bind to one or more pathophysiologically relevant extracellular orcellular structure;3. lead to reduction of the pathophysiologically relevant structure invitro or in vivo;4. lead to reduction of the pathophysiologically relevant structure andto the reduction of a toxicity associated therewith;5. lead to blocking or delaying a disease process;6. lead to regeneration of cellular and organ-specific and organismicfunctions and possibly to a secondary prevention of the recurrence ofthe original pathophysiology after degradation of the toxicity connectedwith the pathophysiologically relevant structure; and/or7. is not associated with increased microhemorrhages

Furthermore, the absence of cross-reactivity with physiologicalprecursors or derivatives leads to the consequence that, firstly, theconcentrations are predictable as sink effects in healthy tissuestructures are avoided and, secondly, that autoimmune responses in thesense of undesired side effects are substantially missing. In addition,previous reports suggested an association of cerebral amyloid angiopathy(CAA) with compromised vascular reactivity in a transgenic mouse modelwith CAA (Mueggler et al., J Neurosci 22 (2002), 7218-24.). The severeCAA occurring in old arc Aβ mice (Knobloch et al., Neurobiol. Aging28:1297-1306 (2007) epub Jul. 31, 2006) might thus constrain thevasodilative flexibility of affected blood vessels. In accordance withthe present invention it is prudent to expect that treatment with theantibodies of the present invention can improve vasoreactivity andcerebral blood flow in aged APP transgenic mice. This may be validatedby using the arc Aβ mice model described in Knobloch et al. (2006),supra, and disclosed in US application “Transgenic animal model forAlzheimer's disease” by Grimm et al., Ser. No. 60/934,291 filed on Jun.11, 2007, the disclosure content of which is incorporated herein byreference.

III. Antibodies

The present invention is further directed to the binding molecules e.g.antibodies and binding fragments, variants, and derivatives thereofshown in Table 2 and 3. The present invention is more specificallydirected to an antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody specifically binds to the sameneoepitope of a disorder-associated protein as a reference antibodyselected from the group consisting of NI-101.10, NI-101.11, NI-101.12,NI-101.13, NI-101.12F6A, NI-101.13A, and NI-101.13B.

The invention is further drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibodycompetitively inhibits a reference antibody selected from the groupconsisting of NI-101.10, NI-101.11, NI-101.12, NI-101.13, NI-101.12F6A,NI-101.13A, and NI-101.13B from binding to the neoepitope of adisorder-associated protein.

The invention is also drawn to an antibody, or antigen-binding fragment,variant or derivatives thereof, where the antibody comprises an antigenbinding domain identical to that of an antibody selected from the groupconsisting of NI-101.10, NI-101.11, NI-101.12, NI-101.13, NI-101.12F6A,NI-101.13A, and NI-101.13B.

The present invention further exemplifies several such bindingmolecules, e.g. antibodies and binding fragments thereof, which may becharacterized by comprising in their variable region, e.g. bindingdomain at least one complementarity determining region (CDR) of the VHand/or VL variable region comprising any one of the amino acid sequencesdepicted in Table 2 (VH) and Table 3 (VL).

TABLE 2Amino acid sequences of the V_(H) region of neoepitope specific antibodies.Antibody Variable heavy chain sequence NI-101.10EVQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGIHWVRQAPGKGLEWVAVIWFDGIKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTT VTVSS(SEQ ID NO: 4) NI-101.11EVQLVQSGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 6) NI-101.12EVQLVESGPGLVKPAETLSLTCTVSGGSIRSGSICWYWIRQPPGKGLEWIGYFCYSGATFYTPSLRGRLTISVDASKNQLSLSLSSVTAADTAVYYCARRAGENSGGIEPYYGMDVWGQGTTVTVSS (SEQ ID NO: 10) NI-101.13QVQLQESGPGLVKPSETLSLTCTVSGGSISRRSYYWGWIRQSPGKGLEWSGSIHYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSRWGSSWVFDYWGQGTLVTVS S (SEQID NO: 14) NI-101.12F6AQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 39) NI-101.13AQVQLQESGPGLVKPSETLSLTCTVSGGSISRRSYYWGWIRQSPGKGLEWSGSIHYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSRWGSSWVFDYWGQGTLVTVSS (SEQ ID NO: 42)NI-101.13BQVQLQESGPGLVKPSETLSLTCTVSGGSISRRSYYWGWIRQSPGKGLEWSGSIHYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSRWGSSWVFDYWGQGTLVTVSS (SEQ ID NO: 43)

TABLE 3Amino acid sequences of the V_(L) region of neoepitope specific antibodies.Antibody Variable light chain sequence (kappa or lambda) NI-101.10EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIKR (SEQ ID NO: 8) NI-101.11EIVLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIKR (SEQ ID NO: 8) NI-101.12DEIVLTQSPSSLSASIGDRVTITCRASESINKYVNWYQQKPGKAPKLLIYAASSLQSGAPSRVSGSGFGRDFSLTISGLQAEDFGAYFCQQSYSAPYTFGQGTKVEIKRT (SEQ ID NO: 12)NI-101.13 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQPPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGYVFGTGTKVTVLG (SEQ ID NO: 16)NI-101.12F6ADIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKR (SEQ ID NO: 41)NI-101.13ADIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTRTFGQGTKVEIKR (SEQ ID NO: 44) NI-101.13BDIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQIPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSRTFGQGTKLEIKR (SEQ ID NO: 45)

The corresponding nucleotide sequences encoding the above-identifiedvariable regions are set forth in the attached sequence listing. Anexemplary set of CDRs of the above amino acid sequences of the VH and/orVL region as depicted in Tables 2 and 3 are given in Table 4. However,as discussed in the following the person skilled in the art is wellaware of the fact that in addition or alternatively CDRs may be used,which differ in their amino acid sequence from those set forth in Table4 by one, two, three or even more amino acids in case of CDR2 and CDR3.

TABLE 4Denomination of CDR protein sequences in Kabat Nomenclature of V_(H) and V_(L)regions of neoepitope specific antibodies. Antibody Variable heavy chainVariable light chain NI-101.10 CDR1 SYGIH (SEQ ID NO: 17)RASQSISSYLN (SEQ ID NO: 23) CDR2 VIWFDGTKKYYTDSVKG (SEQ ID NO: 18)AASSLQS (SEQ ID NO: 24) CDR3 DRGIGARRGPYYMDV (SEQ ID NO: 19)QQSYSTPLT (SEQ ID NO: 25) NI-101.11 CDR1 SYGMH (SEQ ID NO: 20)RASQSISSYLN (SEQ ID NO: 23) CDR2 VIWFDGTKKYYTDSVKG (SEQ ID NO: 21)AASSLQS (SEQ ID NO: 24) CDR3 DRGIGARRGPYYMDV (SEQ ID NO: 22)QQSYSTPLT (SEQ ID NO: 25) NI-101.12 CDR1 SGSIC (SEQ ID NO: 26)RASESINKYVN (SEQ ID NO: 29) CDR2 WIGYFCYSGATFYTPSLRG (SEQ ID NO: 27) AASSLQS (SEQ ID NO: 30) CDR3 RAGENSGGIEPYYGMDV (SEQ ID NO: 28)QQSYSAPYT (SEQ ID NO: 31) NI-101.13 CDR1 RRSYYWG (SEQ ID NO: 32)SGSSSNIGSNYVY (SEQ ID NO: 35) CDR2 SIHYSGSTYYNPSLKS (SEQ ID NO: 33)RNNQRPS (SEQ ID NO: 36) CDR3 SRWGSSWVFDY (SEQ ID NO: 34)AAWDDSLSGYV (SEQ ID NO: 37) NI-101.12F6A CDR1 SYGMH (SEQ ID NO: 20)RASQSISSYLN (SEQ ID NO: 23) CDR2 VIWFDGTKKYYTDSVKG (SEQ ID NO: 21)AASSLQS (SEQ ID NO: 24) CDR3 DRGIGARRGPYYMDV (SEQ ID NO: 22)QQSYSTPLT (SEQ ID NO: 25) NI-101.13A CDR1 RRSYYWG (SEQ ID NO: 32)RASQSISSYLN (SEQ ID NO: 46) CDR2 SIHYSGSTYYNPSLKS (SEQ ID NO: 33)AASSLQS (SEQ ID NO: 47) CDR3 SRWGSSWVFDY (SEQ ID NO: 34)QQSYSTRT (SEQ ID NO: 48) NI-101.13B CDR1 RRSYYWG (SEQ ID NO: 32)RASQSISSWLA (SEQ ID NO: 49) CDR2 SIHYSGSTYYNPSLKS (SEQ ID NO: 33)KASSLES (SEQ ID NO: 50) CDR3 SRWGSSWVFDY (SEQ ID NO: 34)QQYNSYSRT (SEQ ID NO: 51)

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the VH and/or VLregion as depicted in Tables 2 and 3. Alternatively, the antibody of thepresent invention is an antibody or antigen-binding fragment thereof,which competes for binding to the neoeptitope with at least one of theantibodies having the VH and/or VL region as depicted in Tables 2 and 3.Those antibodies may be murine as well, however, humanized, xenogeneic,or chimeric human-murine antibodies being preferred, in particular fortherapeutic applications. An antigen-binding fragment of the antibodycan be, for example, a single chain Fv fragment (scFv), a F(ab′)fragment, a F(ab) fragment, and an F(ab′)₂ fragment. For someapplications only the variable regions of the antibodies are required,which can be obtained by treating the antibody with suitable reagents soas to generate Fab′, Fab, or F(ab″)₂ portions. Such fragments aresufficient for use, for example, in immunodiagnostic proceduresinvolving coupling the immunospecific portions of immunoglobulins todetecting reagents such as radioisotopes.

The present invention is further directed to isolated polypeptides whichmake up the antibodies of the present invention. Antibodies of thepresent invention comprise polypeptides, e.g., amino acid sequencesencoding specific antigen binding regions derived from immunoglobulinmolecules. A polypeptide or amino acid sequence “derived from” adesignated protein refers to the origin of the polypeptide having acertain amino acid sequence. In certain cases, the polypeptide or aminoacid sequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a portion thereof,wherein the portion consists of at least 10-20 amino acids, at least20-30 amino acids, at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe starting sequence.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH), where at least one ofVH-CDRs of the heavy chain variable region or at least two of theVH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or95% identical to reference heavy chain VH-CDR1, VH-CDR2 or VH-CDR3 aminoacid sequences from the antibodies disclosed herein. Alternatively, theVH-CDR1, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%,90% or 95% identical to reference heavy chain VH-CDR1, VH-CDR2 andVH-CDR3 amino acid sequences from the antibodies disclosed herein. Thus,according to this embodiment a heavy chain variable region of theinvention has VH-CDR1, VH-CDR2 and VH-CDR3 polypeptide sequences relatedto the groups shown in Table 4, supra. While Table 4 shows VH-CDRsdefined by the Kabat system, other CDR definitions, e.g., VH-CDRsdefined by the Chothia system, are also included in the presentinvention, and can be easily identified by a person of ordinary skill inthe art using the data presented in Tables 2 and 3.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of or consisting of animmunoglobulin heavy chain variable region (VH) in which the VH-CDR1,VH-CDR2 and VH-CDR3 regions have polypeptide sequences which areidentical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in Table 4.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the VH-CDR1,VH-CDR2 and VH-CDR3 regions have polypeptide sequences which areidentical to the VH-CDR1, VH-CDR2 and VH-CDR3 groups shown in Table 4,except for one, two, three, four, five, or six amino acid substitutionsin any one VH-CDR. In certain embodiments the amino acid substitutionsare conservative.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL), where at least one ofthe VL-CDRs of the light chain variable region or at least two of theVL-CDRs of the light chain variable region are at least 80%, 85%, 90% or95% identical to reference light chain VL-CDR1, VL-CDR2 or VL-CDR3 aminoacid sequences from antibodies disclosed herein. Alternatively, theVL-CDR1, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%,90% or 95% identical to reference light chain VL-CDR1, VL-CDR2 andVL-CDR3 amino acid sequences from antibodies disclosed herein. Thus,according to this embodiment a light chain variable region of theinvention has VL-CDR1, VL-CDR2 and VL-CDR3 polypeptide sequences relatedto the polypeptides shown in Table 4, supra. While Table 4 shows VL-CDRsdefined by the Kabat system, other CDR definitions, e.g., VL-CDRsdefined by the Chothia system, are also included in the presentinvention.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL) in which the VL-CDR1,VL-CDR2 and VL-CDR3 regions have polypeptide sequences which areidentical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in Table 4.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VL) in which the VL-CDR1,VL-CDR2 and VL-CDR3 regions have polypeptide sequences which areidentical to the VL-CDR1, VL-CDR2 and VL-CDR3 groups shown in Table 4,except for one, two, three, four, five, or six amino acid substitutionsin any one VL-CDR. In certain embodiments the amino acid substitutionsare conservative.

An immunoglobulin or its encoding cDNAs may be further modified. Thus,in a further embodiment the method of the present invention comprisesany one of the step(s) of producing a chimeric antibody, humanizedantibody, single-chain antibody, Fab-fragment, bi-specific antibody,fusion antibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodiesare obtained by the phage display technique, surface plasmon resonanceas employed in the BIAcore system can be used to increase the efficiencyof phage antibodies which bind to the same epitope as that of any one ofthe antibodies described herein (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). Theproduction of chimeric antibodies is described, for example, ininternational application WO89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-A10 239 400 and international application WO90/07861. A further source ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human antibodies in mice isdescribed in, e.g., international applications WO91/10741, WO94/02602,WO96/34096 and WO 96/33735. As discussed above, the antibody of theinvention may exist in a variety of forms besides complete antibodies;including, for example, Fv, Fab and F(ab)₂, as well as in single chains;see e.g. international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses small peptides includingthose containing a binding molecule as described above, for examplecontaining the CDR3 region of the variable region of any one of thementioned antibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can be produced by recombinant techniques by incorporatingthe DNA expressing the peptide into an expression vector andtransforming cells with the expression vector to produce the peptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment which is obtainable in accordance withabove described means and display the mentioned properties, i.e. whichspecifically recognize a neoepitope. Such antibodies and bindingmolecules can be tested for their binding specificity and affinity byfor example by using the method of isolating neoepitope specific bindingmolecules described hereinbefore.

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells or B memory cells, the immortalized cells can beused as a source of rearranged heavy chain and light chain loci forsubsequent expression and/or genetic manipulation. Rearranged antibodygenes can be reverse transcribed from appropriate mRNAs to produce cDNA.If desired, the heavy chain constant region can be exchanged for that ofa different isotype or eliminated altogether. The variable regions canbe linked to encode single chain Fv regions. Multiple Fv regions can belinked to confer binding ability to more than one target or chimericheavy and light chain combinations can be employed. Once the geneticmaterial is available, design of analogs as described above which retainboth their ability to bind the de-sired target is straightforward.Methods for the cloning of antibody variable regions and generation ofrecombinant antibodies are known to the person skilled in the art andare described, for example, Gilliland et al., Tissue Antigens 47 (1996),1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

Once the target structure, e.g. the disease-associated protein has beentagged by the sample and respective binding molecule therein it may beidentified by means and methods well known in the art, for example usingmass spectrometric (MS) techniques such as those described ininternational application WO00/11208 and specifically those described inHock et al., Nat Med 8 (2002), 1270-1275; Hock et al., Neuron 38 (2003),547-554. Thus, in case the antibody identified in accordance withpresent invention produced in vitro binds to pathological structures,for example to beta-amyloid plaques in pathological brain area sections,but not significantly to healthy tissues, a promising antibody candidatehas been identified whose molecular target structure can subsequently beenriched and purified via its binding properties to the antibody frompathological tissues and, as a result, can be identified andcharacterized by means of protein analytical and mass spectrometricmethods, like for example MALDI/TOF (Williams, Methods Cell. Biol. 62(2000), 449-453; Yates, J. Mass. Spectrom. 33 (1998), 1-19).

Accordingly, in another embodiment the present invention relates to anantigen which is recognized by the binding molecule, especially antibodyof the present invention described hereinbefore, and which preferably isat least part of a disorder-associated protein.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antigen or binding molecule of the presentinvention, in case of the antibody preferably at least a variable regionof an immunoglobulin chain of the antibody described above. Typically,said variable region encoded by the polynucleotide comprises at leastone complementarity determining region (CDR) of the VH and/or VL of thevariable region of the said antibody. The person skilled in the artknows that each variable domain (the heavy chain VH and light chain VL)of an antibody comprises three hypervariable regions, sometimes calledcomplementarity determining regions or “CDRs” flanked by four relativelyconserved framework regions or “FRs” and refer to the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable regions or CDRs of the human IgG subtype of antibodycomprise amino acid residues from residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain as described by Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)and/or those residues from a hypervariable loop, e.g. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain as described by Chothia et al., J. MoI. Biol. 196 (1987),901-917. Framework or FR residues are those variable domain residuesother than and bracketing the hypervaribale regions. The term “specificbinding” refers to antibody binding to a predetermined antigen.Typically, the antibody binds with a dissociation constant (KD) of 10⁻⁷M or less, and binds to the predetermined antigen with a KD that is atleast twofold less than its KD for binding to a nonspecific antigen(e.g., BSA, casein, or any other specified polypeptide) other than thepredetermined antigen. The phrases “an antibody recognizing an antigen”and “an antibody specific for an antigen” are used interchangeablyherein with the term “an antibody which binds specifically to anantigen”. As used herein “highly specific” binding means that therelative KD of the antibody for the specific target epitope, e.g.neoepitope is at least 10-fold less than the KD for binding thatantibody to other ligands or to the native counterpart of thedisease-associated protein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method; see, for example, Berzofsky etal., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W.E., Ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W.H. Freeman and Company New York, N.Y. (1992), and methods describedherein. General techniques for measuring the affinity of an antibody foran antigen include ELISA, RIA, and surface plasmon resonance. Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art willreadily appreciate that using the variable domains or CDRs describedherein antibodies can be constructed according to methods known in theart, e.g., as described in European patent applications EP 0 451 216 A1and EP 0 549 581 A1. Furthermore, the person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat. Thus, the present invention also relates to antibodieswherein one or more of the mentioned CDRs comprise one or more,preferably not more than two amino acid substitutions. Preferably, theantibody of the invention comprises in one or both of its immunoglobulinchains two or all three CDRs of the variable regions as set forth inTable 4.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the invention include an antibody,or antigen-binding fragment, variant, or derivative thereof, in which atleast a fraction of one or more of the constant region domains has beendeleted or otherwise altered so as to provide desired biochemicalcharacteristics such as reduced effector functions, the ability tonon-covalently dimerize, increased ability to localize at the site of atumor, reduced serum half-life, or increased serum half-life whencompared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted. Inother embodiments, certain antibodies for use in the diagnostic andtreatment methods described herein have a constant region, e.g., an IgGheavy chain constant region, which is altered to eliminateglycosylation, referred to elsewhere herein as aglycosylated or “agly”antibodies. Such “agly” antibodies may be prepared enzymatically as wellas by engineering the consensus glycosylation site(s) in the constantregion. While not being bound by theory, it is believed that “agly”antibodies may have an improved safety and stability profile in vivo.Methods of producing aglycosylated antibodies, having desired effectorfunction are found for example in WO 2005/018572, which is incorporatedby reference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein.

In certain embodiments both the variable and constant regions of theantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention are fully human. Fully human antibodies can bemade using techniques that are known in the art and as described herein.For example, fully human antibodies against a specific antigen can beprepared by administering the antigen to a transgenic animal which hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled. Exemplarytechniques that can be used to make such antibodies are described inU.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Other techniques areknown in the art. Fully human antibodies can likewise be produced byvarious display technologies, e.g., phage display or other viral displaysystems, as described in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be made or manufactured using techniquesthat are known in the art. In certain embodiments, antibody molecules orfragments thereof are “recombinantly produced,” i.e., are produced usingrecombinant DNA technology. Exemplary techniques for making antibodymolecules or fragments thereof are discussed in more detail elsewhereherein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention will not elicit adeleterious immune response in the animal to be treated, e.g., in ahuman. In certain embodiments, binding molecules, e.g., antibodies, orantigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, VH and VL sequences from the startingantibody are analyzed and a human T cell epitope “map” from each Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative VH and VL sequences are designed comprising combinations ofamino acid substitutions and these sequences are subsequentlyincorporated into a range of binding polypeptides, e.g.,neo-epitope-specific antibodies or immunospecific fragments thereof foruse in the diagnostic and treatment methods disclosed herein, which arethen tested for function. Typically, between 12 and 24 variantantibodies are generated and tested. Complete heavy and light chaingenes comprising modified V and human C regions are then cloned intoexpression vectors and the subsequent plasmids introduced into celllines for the production of whole antibody. The antibodies are thencompared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art. In certain embodiments,antibodies of the present invention are derived from human B cells whichhave been immortalized via transformation with Epstein-Barr virus, asdescribed herein.

In the well known hybridoma process (Kohler et al., Nature 256:495(1975)) the relatively short-lived, or mortal, lymphocytes from amammal, e.g., B cells derived from a human subject as described herein,are fused with an immortal tumor cell line (e.g.,. a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and regrowth with each individual strain comprisingspecific genes for the formation of a single antibody. They produceantibodies, which are homogeneous against a desired antigen and, inreference to their pure genetic parentage, are termed “monoclonal.”

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA), or neoepitope binding assays as describedherein. After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,pp 59-103 (1986)). It will further be appreciated that the monoclonalantibodies secreted by the subclones may be separated from culturemedium, ascites fluid or serum by conventional purification proceduressuch as, for example, protein-A, hydroxylapatite chromatography, gelelectrophoresis, dialysis or affinity chromatography.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain.

Completely human antibodies, such as described herein, are particularlydesirable for therapeutic treatment of human patients. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety. Human antibodies ofthe present invention are isolated, e.g., from patients who are symptomfree but affected with the risk of developing a disorder, e.g.,Alzheimer's disease, or a patient with the disorder but with anunusually stable disease course.

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as, but not limited to, E. coli cells, simian COS cells, ChineseHamster Ovary (CHO) cells or myeloma cells that do not otherwise produceimmunoglobulins. More particularly, the isolated DNA (which may besynthetic as described herein) may be used to clone constant andvariable region sequences for the manufacture antibodies as described inNewman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which isincorporated by reference herein. Essentially, this entails extractionof RNA from the selected cells, conversion to cDNA, and amplification byPCR using Ig specific primers. Suitable primers for this purpose arealso described in U.S. Pat. No. 5,658,570. As will be discussed in moredetail below, transformed cells expressing the desired antibody may begrown up in relatively large quantities to provide clinical andcommercial supplies of the immunoglobulin.

In one embodiment, an antibody of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,an antibody of the invention comprises at least two CDRs from one ormore antibody molecules. In another embodiment, an antibody of theinvention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least four CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least five CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least six CDRs from one or more antibodymolecules. Exemplary antibody molecules comprising at least one CDR thatcan be included in the subject antibodies are described herein.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) fora listing of human framework regions). In certain embodiments, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds to at least one epitopeof a desired polypeptide. In certain embodiments, one or more amino acidsubstitutions may be made within the framework regions, to, e.g.,improve binding of the antibody to its antigen. Additionally, suchmethods may be used to make amino acid substitutions or deletions of oneor more variable region cysteine residues participating in an intrachaindisulfide bond to generate antibody molecules lacking one or moreintrachain disulfide bonds. Other alterations to the polynucleotide areencompassed by the present invention and within the skill of the art.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-554 (1989)) can be adapted to produce singlechain antibodies. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain antibody. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the VH and VL genes can be amplifiedusing, e.g., RT-PCR. The VH and VL genes can be cloned into an antibodyexpression vector and transfected into cells (e.g., eukaryotic orprokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔCH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgG1 human constantdomain (see, e.g., WO 02/060955A2 and WO02/096948A2). This vector isengineered to delete the CH2 domain and provide a synthetic vectorexpressing a domain deleted IgG1 constant region.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art(see, e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1).

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement binding) to be modulated.Such partial deletions of the constant regions may improve selectedcharacteristics of the antibody (serum half-life) while leaving otherdesirable functions associated with the subject constant region domainintact. Moreover, as alluded to above, the constant regions of thedisclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it may be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa disorder-associated polypeptide or fragment or variant thereof.Standard techniques known to those of skill in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody,including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH region, VH-CDR1, VH-CDR2, VH-CDR3, VLregion, VL-CDR1, VL-CDR2, or VL-CDR3. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a side chain with a similar charge. Familiesof amino acid residues having side chains with similar charges have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind an disorder-associated polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral missense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen binding activity oralteration in binding activity (e.g, improvements in antigen bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of a disorder-associatedpolypeptide) can be determined using techniques described herein or byroutinely modifying techniques known in the art.

IV. Polynucleotides Encoding Antibodies

In accordance with the above, the present invention also relates to apolynucleotide encoding a binding molecule of the present invention,e.g., an antibody. In case of the antibody the polynucleotide may encodeat least a variable region of an immunoglobulin chain of the antibodydescribed above. The polynucleotide of the invention encoding the abovedescribed antibody may be, e.g., DNA, cDNA, RNA or syntheticallyproduced DNA or RNA or a recombinantly produced chimeric nucleic acidmolecule comprising any of those polynucleotides either alone or incombination. Preferably said polynucleotide is part of a vector. Suchvectors may comprise further genes such as marker genes which allow forthe selection of said vector in a suitable host cell and under suitableconditions. Preferably, the polynucleotide of the invention isoperatively linked to expression control sequences allowing expressionin prokaryotic or eukaryotic cells. Expression of said polynucleotidecomprises transcription of the polynucleotide into a translatable mRNA.Regulatory elements ensuring expression in eukaryotic cells, preferablymammalian cells, are well known to those skilled in the art. Theyusually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers,and/or naturally associated or heterologous promoter regions.

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxyribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe antibody may be made, either simultaneously or separately, usingreverse transcriptase and DNA polymerase in accordance with well knownmethods. PCR may be initiated by consensus constant region primers or bymore specific primers based on the published heavy and light chain DNAand amino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at airy point during the isolation process or subsequentanalysis.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(VH), where at least one of the CDRs of the heavy chain variable regionor at least two of the VH-CDRs of the heavy chain variable region are atleast 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDR1,VH-CDR2, or VH-CDR3 amino acid sequences from the antibodies disclosedherein. Alternatively, the VH-CDR1, VH-CDR2, and VH-CDR3 regions of theVH are at least 80%, 85%, 90% or 95% identical to reference heavy chainVH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences from the antibodiesdisclosed herein. Thus, according to this embodiment a heavy chainvariable region of the invention has VH-CDR1, VH-CDR2, or VH-CDR3polypeptide sequences related to the polypeptide sequences shown inTable 4.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(VL), where at least one of the VL-CDRs of the light chain variableregion or at least two of the VL-CDRs of the light chain variable regionare at least 80%, 85%, 90% or 95% identical to reference light chainVL-CDR1, VL-CDR2, or VL-CDR3 amino acid sequences from the antibodiesdisclosed herein. Alternatively, the VL-CDR1, VL-CDR2, and VL-CDR3regions of the VL are at least 80%, 85%, 90% or 95% identical toreference light chain VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Thus, according to this embodimenta light chain variable region of the invention has VL-CDR1, VL-CDR2, orVL-CDR3 polypeptide sequences related to the polypeptide sequences shownin Table 4.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VH)in which the VH-CDR1, VH-CDR2, and VH-CDR3 regions have polypeptidesequences which are identical to the VH-CDR1, VH-CDR2, and VH-CDR3groups shown in Table 4.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

TABLE 5Polynucleotide sequences of the V_(H) region of neoepitope specific antibodies.Antibody Variable heavy chain sequence NI-101.10GAGGTGCAGCTAGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC (SEQ IDTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATACACTGGGTCCGCCAGGCTCCAGGCA NO: 3)AGGGGCTGGAGTGGGTGGCAGTTATATGGTTTGATGGAACTAAAAAATACTATACAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGGTATAGGAGCTCGGCGGGGGCCGTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA NI-101.11GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC (SEQ IDTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCA NO: 56)AGGGGCTGGAGTGGGTGGCAGTTATATGGTTTGATGGAACTAAAAAATACTATACAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGGTATAGGAGCTCGGCGGGGGCCGTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA NI-101.11GAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAGCCCGGCCGGAGCCTGCGGCTGAG (SEQ IDCTGCGCCGCCAGCGGCTTCGCCTTCAGCAGCTACGGCATGCACTGGGTGCGGCAGGCCCCCGG NO: 5)CAAGGGCCTGGAGTGGGTGGCCGTGATCTGGTTCGACGGCACCAAGAAGTACTACACCGACAGC (codon-GTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAoptimized)CCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGGACCGGGGCATCGGCGCCCGGCGGGGCCCCTACTACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGC NI-101.12GAGGTGCAGCTGGTGGAGAGCGGCCCCGGCCTGGTGAAGCCCGCCGAGACCCTGAGCCTGACC (SEQ IDTGCACCGTGAGCGGCGGCAGCATCCGGAGCGGCAGCATCTGCTGGTACTGGATCCGGCAGCCC NO: 9)CCCGGCAAGGGCCTGGAGTGGATCGGCTACTTCTGCTACAGCGGCGCCACCTTCTACACCCCCAGCCTGCGGGGCCGGCTGACCATCAGCGTGGACGCCAGCAAGAACCAGCTGAGCCTGAGCCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGGCGGGCCGGCGAGAACAGCGGCGGCATCGAGCCCTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGC AGCNI-101.13CAGGTACAGCTGCAGGAGTCAGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCT (SEQ IDGCACTGTCTCTGGTGGCTCCATCAGCAGAAGAAGTTACTACTGGGGCTGGATCCGCCAGTCCCC NO: 13)AGGGAAGGGGCTGGAGTGGAGTGGAAGTATCCATTATAGCGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCTGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTTACCGCCGCAGACACGGCTGTCTATTACTGTGCGAGATCACGTTGGGGCAGCAGCTGGGTATTTGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCTTCG NI-101.12F6ACAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC (SEQ IDTGTGCAGCGTCTGGATTCGCCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCA NO: 38)AGGGGCTGGAGTGGGTGGCAGTTATATGGTTTGATGGAACTAAAAAATACTATACAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGGTATAGGAGCTCGGCGGGGGCCGTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA NI-101.13ACAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCT (SEQ IDGCACTGTCTCTGGTGGCTCCATCAGCAGAAGAAGTTACTACTGGGGCTGGATCCGCCAGTCCCC NO: 52)AGGGAAGGGGCTGGAGTGGAGTGGAAGTATCCATTATAGCGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCTGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTTACCGCCGCAGACACGGCTGTCTATTACTGTGCGAGATCACGTTGGGGCAGCAGCTGGGTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG NI-101.13BCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCT (SEQ IDGCACTGTCTCTGGTGGCTCCATCAGCAGAAGAAGTTACTACTGGGGCTGGATCCGCCAGTCCCC NO: 53)AGGGAAGGGGCTGGAGTGGAGTGGAAGTATCCATTATAGCGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCTGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTTACCGCCGCAGACACGGCTGTCTATTACTGTGCGAGATCACGTTGGGGCAGCAGCTGGGTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

TABLE 6Polynucleotide sequences of the V_(L) region of neoepitope specific antibodies.Antibody Variable light chain sequence (kappa or lambda) NI-101.10GAAATTGTGCTGACTCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT andTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCNI-101.11TAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTG(SEQ IDGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTNO: 7) GTCAGCAGAGTTACAGTACCCCTCTCACTTTCGGCGGAGGGACCAAGCTCGAGATCAAACGTACG NI-101.12GACGAGATCGTGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCATCGGCGACCGGGTGACC (SEQ IDATCACCTGCCGGGCCAGCGAGAGCATCAACAAGTACGTGAACTGGTACCAGCAGAAGCCCGGCA NO: 11)AGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGCCCCCAGCCGGGTGAGCGGCAGCGGCTTCGGCCGGGACTTCAGCCTGACCATCAGCGGCCTGCAGGCCGAGGACTTCGGCGCCTACTTCTGCCAGCAGAGCTACAGCGCCCCCTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGACC NI-101.13CAGAGCGTGCTGACCCAGCCGCCGAGCGCGAGCGGCACCCCGGGCCAGCGCGTGACCATTAGC (SEQ IDTGCAGCGGCAGCAGCAGCAACATTGGCAGCAACTATGTGTATTGGTATCAGCAGCCGCCGGGCA NO: 15)CCGCGCCGAAACTGCTGATTTATCGCAACAACCAGCGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGCAGCAAAAGCGGCACCAGCGCGAGCCTGGCGATTAGCGGCCTGCGCAGCGAAGATGAAGCGGATTATTATTGCGCGGCGTGGGATGATAGCCTGAGCGGCTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTG NI-101.12F6AGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC(SEQ IDTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCNO: 40)CTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAGCAGAGTTACAGTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTNI-101.13AGACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC(SEQ IDTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCNO: 54)CTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCAGAACGTTCGGCCAAGGGACCAAGGTGGAGATCAAACGTACGNI-101.13BGACATCCAGTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAC(SEQ ID TTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGATTCCAGGGAAAGCCNO: 55)CCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTCGAACGTTCGGCCAAGGGACCAAGCTGGAGATCAAACGTA CG

In this respect, the person skilled in the art will readily appreciatethat the polynucleotides encoding at least the variable domain of thelight and/or heavy chain may encode the variable domains of bothimmunoglobulin chains or only one. Likewise, said polynucleotides may beunder the control of the same promoter or may be separately controlledfor expression. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoterin E. coli, and examples for regulatory elements permitting expressionin eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or theCMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or a globinintron in mammalian and other animal cells. Beside elements which areresponsible for the initiation of transcription such regulatory elementsmay also comprise transcription termination signals, such as theSV40-poly-A site or the tk-poly-A site, downstream of thepolynucleotide. Furthermore, depending on the expression system usedleader sequences capable of directing the polypeptide to a cellularcompartment or secreting it into the medium may be added to the codingsequence of the polynucleotide of the invention and are well known inthe art. The leader sequence(s) is (are) assembled in appropriate phasewith translation, initiation and termination sequences, and preferably,a leader sequence capable of directing secretion of translated protein,or a portion thereof, into the periplasmic space or extracellularmedium. Optionally, the heterologous sequence can encode a fusionprotein including a C- or N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product. In this context, suitable expressionvectors are known in the art such as Okayama-Berg cDNA expression vectorpcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), orpSPORT1 (GIBCO BRL). Preferably, the expression control sequences willbe eukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences, and, as desired, the collection and purification of theimmunoglobulin light chains, heavy chains, light/heavy chain dimers orintact antibodies, binding fragments or other immunoglobulin forms mayfollow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press,N.Y., (1979).

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeiera al., BioTechniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably poly A+RNA, isolated from, any tissue or cellsexpressing the neoantigen-specific antibody, such as hybridoma cellsselected to express an antibody) by PCR amplification using syntheticprimers hybridizable to the 3′ and 5′ ends of the sequence or by cloningusing an oligonucleotide probe specific for the particular gene sequenceto identify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

V. Expression of Antibody Polypeptides

The present invention also involves a method for producing cells capableof expressing an antibody of the invention or its correspondingimmunoglobulin chain(s) comprising genetically engineering cells withthe polynucleotide or with the vector of the invention. The cellsobtainable by the method of the invention can be used, for example, totest the interaction of the antibody of the invention with its antigen.

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule described herein. Once a polynucleotide encoding anantibody molecule or a heavy or light chain of an antibody, or portionthereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The present invention relates to vectors, particularly plasmids,cosmids, viruses and bacteriophages used conventionally in geneticengineering that comprise a polynucleotide encoding the antigen orpreferably a variable domain of an immunoglobulin chain of an antibodyof the invention; optionally in combination with a polynucleotide of theinvention that encodes the variable domain of the other immunoglobulinchain of the antibody of the in-vention. Preferably, said vector is anexpression vector and/or a gene transfer or targeting vector. Expressionvectors derived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotides or vector of the inventioninto targeted cell population. Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectors;see, for example, the techniques described in Sambrook, MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Alternatively, thepolynucleotides and vectors of the invention can be reconstituted intoliposomes for delivery to target cells. The vectors containing thepolynucleotides of the invention (e.g., the heavy and/or light variabledomain(s) of the immunoglobulin chains encoding sequences and expressioncontrol sequences) can be transferred into the host cell by well knownmethods, which vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts; see Sambrook, supra.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthetic as discussedabove. In one embodiment, this is effected using a proprietaryexpression vector of Biogen IDEC, Inc., referred to as NEOSPLA(disclosed in U.S. Pat. No. 6,159,730). This vector contains thecytomegalovirus promoter/enhancer, the mouse beta globin major promoter,the SV40 origin of replication, the bovine growth hormonepolyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2,the dihydrofolate reductase gene and leader sequence. This vector hasbeen found to result in very high level expression of antibodies uponincorporation of variable and constant region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification. Of course, any expression vector which is capable ofeliciting expression in eukaryotic cells may be used in the presentinvention. Examples of suitable vectors include, but are not limited toplasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2,pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (availablefrom Invitrogen, San Diego, Calif.), and plasmid pCI (available fromPromega, Madison, Wis.). In general, screening large numbers oftransformed cells for those which express suitably high levels ifimmunoglobulin heavy and light chains is routine experimentation whichcan be carried out, for example, by robotic systems. Vector systems arealso taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which isincorporated by reference in its entirety herein. This system providesfor high expression levels, e.g., >30 pg/cell/day. Other exemplaryvector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

In other preferred embodiments the antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed inUnited States Patent Application Publication No. 2003-0157641 A1, filedNov. 18, 2002 and incorporated herein in its entirety. In these novelexpression systems, multiple gene products of interest such as heavy andlight chains of antibodies may be produced from a single polycistronicconstruct. These systems advantageously use an internal ribosome entrysite (IRES) to provide relatively high levels of antibodies. CompatibleIRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is alsoincorporated herein. Those skilled in the art will appreciate that suchexpression systems may be used to effectively produce the full range ofantibodies disclosed in the instant application.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection (including electrophoresis and electroporation), protoplastfusion, calcium phosphate precipitation, cell fusion with enveloped DNA,microinjection, and infection with intact virus. See, Ridgway, A. A. G.“Mammalian Expression Vectors” Vectors, Rodriguez and Denhardt, Eds.,Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988).Typically, plasmid introduction into the host is via electroporation.The host cells harboring the expression construct are grown underconditions appropriate to the production of the light chains and heavychains, and assayed for heavy and/or light chain protein synthesis.Exemplary assay techniques include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorteranalysis (FACS), immunohistochemistry and the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

The present invention furthermore relates to host cells transformed witha polynucleotide or vector of the invention. Said host cell may be aprokaryotic or eukaryotic cell. The polynucleotide or vector of theinvention which is present in the host cell may either be integratedinto the genome of the host cell or it may be maintainedextrachromosomally. The host cell can be any prokaryotic or eukaryoticcell, such as a bacterial, insect, fungal, plant, animal or human cell.Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecules for theexpression of an antibody of the invention or the correspondingimmu-noglobulin chains. Prokaryotic hosts may include gram negative aswell as gram positive bacteria such as, for example, E. coli, S.typhimurium, Serratia marcescens and Bacillus subtilis. The term“eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells, most preferably HEK 293, NSO and CHO cells.Depending upon the host employed in a recombinant production procedure,the antibodies or immunoglobulin chains encoded by the polynucleotide ofthe present invention may be glycosylated or may be non-glycosylated.Antibodies of the invention or the corresponding immunoglobulin chainsmay also include an initial methionine amino acid residue. Apolynucleotide of the invention can be used to transform or transfectthe host using any of the techniques commonly known to those of ordinaryskill in the art. Furthermore, methods for preparing fused, operablylinked genes and expressing them in, e.g., mammalian cells and bacteriaare well-known in the art (Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).The genetic constructs and methods described therein can be utilized forexpression of the antibody of the invention or the correspondingimmunoglobulin chains in eukaryotic or prokaryotic hosts. In general,expression vectors containing promoter sequences which facilitate theefficient transcription of the inserted polynucleotide are used inconnection with the host. The expression vector typically contains anorigin of replication, a promoter, and a terminator, as well as specificgenes which are capable of providing phenotypic selection of thetransformed cells. Suitable source cells for the DNA sequences and hostcells for immunoglobulin expression and secretion can be obtained from anumber of sources, such as the American Type Culture Collection(“Catalogue of Cell Lines and Hybridomas,” Fifth edition (1985)Rockville, Md., U.S.A., which is incorporated herein by reference).Furthermore, transgenic animals, preferably mammals, comprising cells ofthe invention may be used for the large scale production of the antibodyof the invention.

Thus, in a further embodiment, the present invention relates to a methodfor the production of a disorder-associated protein specific bindingmolecule, e.g., an antibody or a binding fragment or immunoglobulinchain(s) thereof, said method comprising

(a) culturing a cell as described above; and

-   (b) isolating said antigen, binding molecule, antibody or binding    fragment or immunoglobulin chain(s) thereof from the culture.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Onceexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) of the invention can then be isolated from thegrowth medium, cellular lysates, or cellular membrane fractions. Theisolation and purification of the, e.g., recombinantly expressedantibodies or immunoglobulin chains of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies directed, e.g., against theconstant region of the antibody of the invention. It will be apparent tothose skilled in the art that the antibodies of the invention can befurther coupled to other moieties for, e.g., drug targeting and imagingapplications. Such coupling may be conducted chemically after expressionof the antibody or antigen to site of attachment or the coupling productmay be engineered into the antibody or antigen of the invention at theDNA level. The DNAs are then expressed in a suitable host system, andthe expressed proteins are collected and renatured, if necessary.

Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the antibodies may then be used therapeutically(including extracorporally) or in developing and performing assayprocedures.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77:2197 (1980)). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO cells are particularly preferred. Host celllines are typically available from commercial services, the AmericanTissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can also be expressed non-mammaliancells such as bacteria or insect or yeast or plant cells. Bacteria whichreadily take up nucleic acids include members of the enterobacteriaceae,such as strains of Escherichia coli or Salmonella; Bacillaceae, such asBacillus subtilis; Pneumococcus; Streptococcus, and Haemophilusinfluenzae. It will further be appreciated that, when expressed inbacteria, the heterologous polypeptides typically become part ofinclusion bodies. The heterologous polypeptides must be isolated,purified and then assembled into functional molecules. Where tetravalentforms of antibodies are desired, the subunits will then self-assembleinto tetravalent antibodies (WO02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). Thepresence of the trpl lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in US 2002 0123057 A1.

VI. Fusion Proteins and Conjugates

The antibodies of the present invention can comprise a further domain,said domain being linked by covalent or non-covalent bonds. The linkagecan be based on genetic fusion according to the methods known in the artand described above or can be performed by, e.g., chemical cross-linkingas described in, e.g., international application WO94/04686. Theadditional domain present in the fusion protein comprising the antibodyof the invention may preferably be linked by a flexible linker,advantageously a polypeptide linker, wherein said polypeptide linkercomprises plural, hydrophilic, peptide-bonded amino acids of a lengthsufficient to span the distance between the C-terminal end of saidfurther domain and the N-terminal end of the antibody of the inventionor vice versa. The therapeutically or diagnostically active agent can becoupled to the antibody of the invention or an antigen-binding fragmentthereof by various means. This includes, for example, single-chainfusion proteins comprising the variable regions of the antibody of theinvention coupled by covalent methods, such as peptide linkages, to thetherapeutically or diagnostically active agent. Further examples includemolecules which comprise at least an antigen-binding fragment coupled toadditional molecules covalently or non-covalently include those in thefollowing non-limiting illustrative list. Traunecker, Int. J. CancerSurp. SuDP 7 (1992), 51-52, describe the bispecific reagent janusin inwhich the Fv region directed to CD3 is coupled to soluble CD4 or toother ligands such as OVCA and IL-7. Similarly, the variable regions ofthe antibody of the invention can be constructed into Fv molecules andcoupled to alternative ligands such as those illustrated in the citedarticle. Higgins, J. Infect Disease 166 (1992), 198-202, described ahetero-conjugate antibody composed of OKT3 cross-linked to an antibodydirected to a specific sequence in the V3 region of GP 120. Suchhetero-conjugate antibodies can also be constructed using at least thevariable regions contained in the antibody of the invention methods.Additional examples of specific antibodies include those described byFanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev.Immunol. 12 (1992), 101-124.

In a further embodiment of the present invention, the binding molecule,antibody, immunoglobulin chain or a binding fragment thereof or theantigen is detectably labeled. Labeling agents can be coupled eitherdirectly or indirectly to the antibodies or antigens of the invention.One example of indirect coupling is by use of a spacer moiety.

Hence, the biological activity of the binding molecules, e.g. antibodiesidentified here suggests that they have sufficient affinity to make thempotential candidates for drug localization to cells expressing theappropriate surface structures of the diseased cell and tissue,respectively. This targeting and binding to cells could be useful forthe delivery of therapeutically or diagnostically active agents and genetherapy/gene delivery. Molecules/particles with an antibody of theinvention would bind specifically to cells/tissues expressing thevariant form of the pathological protein, and therefore could havediagnostic and therapeutic use. Thus, the binding molecule, e.g.,antibody or antigen binding fragment thereof of the present inventioncan be labeled (e.g., fluorescent, radioactive, enzyme, nuclearmagnetic, heavy metal) and used to detect specific targets in vivo or invitro including “immunochemistry” like assays in vitro. In vivo theycould be used in a manner similar to nuclear medicine imaging techniquesto detect tissues, cells, or other material expressing the neoepitope.Thus, in a further embodiment the present invention relates to the useof a binding molecule or an antibody of the present invention or bindingfragment thereof for the preparation of a composition for in vivodetection of or targeting a therapeutic and/or diagnostic agent to adisorder-associated protein in the brain, detecting, suppressingformation of or reducing pathological protein aggregates orconformations in a subject, for improving cognition or slowing orreversing cognitive decline associated with diseases, or forextra-corporal extraction of pathological compounds or their precursorsfrom body fluids.

In certain embodiments, antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label).

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinantigen-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species.

As discussed in more detail elsewhere herein, antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, antibodies may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, e.g., Proteins—Structure And Molecular Properties, T. E.Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993);Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of the VHregions of an antibody of the invention or the amino acid sequence ofany one or more of the VL regions of an antibody of the invention orfragments or variants thereof, and a heterologous polypeptide sequence.In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the VH-CDRs of an antibody, or fragments, variants, orderivatives thereof, or the amino acid sequence of any one, two, threeof the VL-CDRs of an antibody, or fragments, variants, or derivativesthereof, and a heterologous polypeptide sequence. In one embodiment, thefusion protein comprises a polypeptide having the amino acid sequence ofa VH-CDR3 of an antibody of the present invention, or fragment,derivative, or variant thereof, and a heterologous polypeptide sequence,which fusion protein specifically binds to at least one neoepitope of adisorder-associated protein. In another embodiment, a fusion proteincomprises a polypeptide having the amino acid sequence of at least oneVH region of an antibody of the invention and the amino acid sequence ofat least one VL region of an antibody of the invention or fragments,derivatives or variants thereof, and a heterologous polypeptidesequence. Preferably, the VH and VL regions of the fusion proteincorrespond to a single source antibody (or scFv or Fab fragment) whichspecifically binds at least one neoepitope of a disorder-associatedprotein. In yet another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises apolypeptide having the amino acid sequence of any one, two, three ormore of the VH CDRs of an antibody and the amino acid sequence of anyone, two, three or more of the VL CDRs of an antibody, or fragments orvariants thereof, and a heterologous polypeptide sequence. Preferably,two, three, four, five, six, or more of the VH-CDR(s) or VL-CDR(s)correspond to single source antibody (or scFv or Fab fragment) of theinvention. Nucleic acid molecules encoding these fusion proteins arealso encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo. Leong, S. R., etal., Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); orWeir et al., Biochem. Soc. Transactions 30:512 (2002).

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed.

In particular, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

The above described fusion protein may further comprise a cleavablelinker or cleavage site for proteinases. These spacer moieties, in turn,can be either insoluble or soluble (Diener et al., Science 231 (1986),148) and can be selected to enable drug release from the antigen at thetarget site. Examples of therapeutic agents which can be coupled to theantibodies and antigens of the present invention for immunotherapy aredrugs, radioisotopes, lectins, and toxins. The drugs with which can beconjugated to the antibodies and antigens of the present inventioninclude compounds which are classically referred to as drugs such asmitomycin C, daunorubicin, and vinblastine. In using radioisotopicallyconjugated antibodies or antigens of the invention for, e.g.,immunotherapy, certain isotopes may be more preferable than othersdepending on such factors as leukocyte distribution as well as stabilityand emission. Depending on the autoimmune response, some emitters may bepreferable to others. In general, α and β particle emittingradioisotopes are preferred in immunotherapy. Preferred are short range,high energy a emitters such as ²¹²Bi. Examples of radioisotopes whichcan be bound to the antibodies or antigens of the invention fortherapeutic purposes include, but are not limited to ¹²⁵I, ¹³¹ I, ⁹⁰Y,⁶⁷Cu, ⁶⁴Y, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. Other therapeuticagents which can be coupled to the binding molecule, e.g., antibody orantigen binding fragment thereof of the invention, as well as ex vivoand in vivo therapeutic protocols, are known, or can be easilyascertained, by those of ordinary skill in the art. Wherever appropriatethe person skilled in the art may use a polynucleotide of the inventionencoding any one of the above described antibodies, antigens or thecorresponding vectors instead of the proteinaeous material itself.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting a binding polypeptide with an activated ester of biotin suchas the biotin N-hydroxysuccinimide ester. Similarly, conjugates with afluorescent marker may be prepared in the presence of a coupling agent,e.g. those listed herein, or by reaction with an isothiocyanate,preferably fluorescein-isothiocyanate. Conjugates of the antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof a neurological disease as part of a clinical testing procedure to,e.g., determine the efficacy of a given treatment and/or preventionregimen. Detection can be facilitated by coupling the antibody, orantigen-binding fragment, variant, or derivative thereof to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,(1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin,Tokyo (1981). The enzyme, which is bound to the antibody will react withan appropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas 152Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

In certain embodiments, a moiety that enhances the stability or efficacyof a binding molecule, e.g., a binding polypeptide, e.g., an antibody orimmunospecific fragment thereof can be conjugated. For example, in oneembodiment, PEG can be conjugated to the binding molecules of theinvention to increase their half-life in vivo. Leong, S. R., et al.,Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weiret al., Biochem. Soc. Transactions 30:512 (2002).

VII. Compositions and Methods of Use

Moreover, the present invention relates to compositions comprising theaforementioned binding molecule, e.g., antibody or antigen bindingfragment thereof of the present invention or chemical derivativesthereof, or the polynucleotide, vector or cell of the invention. Thecomposition of the present invention may further comprise apharmaceutically acceptable carrier. The term “chemical derivative”describes a molecule that contains additional chemical moieties that arenot normally a part of the base molecule. Such moieties may improve thesolubility, half-life, absorption, etc. of the base molecule.Alternatively the moieties may attenuate undesirable side effects of thebase molecule or decrease the toxicity of the base molecule.Furthermore, the pharmaceutical composition of the present invention maycomprise further agents such as interleukins or interferons depending onthe intended use of the pharmaceutical composition. For example, for usein the treatment of Alzheimer's disease the additional agent mayselected from the group consisting of small organic molecules,anti-Abeta antibodies, and combinations thereof. Hence, in a particularpreferred embodiment the present invention relates to the use of thebinding molecule, e.g., antibody or antigen binding fragment thereof ofthe present invention or of a binding molecule having substantially thesame binding specificities of any one thereof, the polynucleotide, thevector or the cell of the present invention for the preparation of apharmaceutical or diagnostic composition for treating or preventing theprogression of Alzheimer's disease; for the amelioration of symptomsassociated with Alzheimer's disease; for diagnosing or screening asubject for the presence of Alzheimer's disease or for determining asubject's risk for developing Alzheimer's disease. Said pharmaceuticalcomposition can be designed to be administered intravenously,intramuscularly, subcutaneously, intraperitoneally, intranasally,parenterally or as an aerosol; see also infra.

Hence, in one embodiment the present invention relates to a method oftreating a neurological disorder characterized by abnormal accumulationand/or deposition of a protein in the central nervous system, whichmethod comprises administering to a subject in need thereof atherapeutically effective amount of any one of the afore-describedbinding molecules, antibodies, antigens, polynucleotides, vectors orcells of the instant invention. The term “neurological disorder”includes but is not limited to Alzheimer's Disease, mild cognitiveimpairment, fronto-temporal dementia, Lewy-body disease, Parkinson'sdisease, Pick's disease, Binswanger's disease; congophilic amyloidangiopathy, cerebral amyloid angiopathy, Down's syndrome, multi-infarctdementia, Huntington's Disease, Creutzfeldt-Jakob Disease, AIDS dementiacomplex, depression, anxiety disorder, phobia, Bell's Palsy, epilepsy,encephalitis, multiple sclerosis; neuromuscular disorders,neurooncological disorders, brain tumors, neurovascular disordersincluding stroke, neuroimmunological disorders, neurootological disease,neurotrauma including spinal cord injury, pain including neuropathicpain, pediatric neurological and neuropsychiatric disorders, sleepdisorders, Tourette syndrome, mild cognitive impairment, vasculardementia, multi-infarct dementia, cystic fibrosis, Gaucher's diseaseother movement disorders and disease of the central nervous system (CNS)in general. Unless stated otherwise, the terms neurodegenerative,neurological or neuropsychiatric are used interchangeably herein.

In the sense of the present invention, a method is disclosed forcharacterizing human antibodies for a large number of diseases and toalso produce said antibodies subsequently in order to employ themdiagnostically, therapeutically, or preventively in such patients whoseimmune system did not react with a corresponding immune response to thedevelopment of the pathology of the disease. In particular, this willhave to be expected in diseases occurring at an advanced age because, asis known, the reactivity of the immune system continuously andsignificantly decreases as the age increases. In these cases,therapeutically or preventively active antibodies could compensate theage-related restrictions of the immune system with respect to blockingthe enrichment of endogenous pathophysiological protein variants andcould thus contribute to a better state of health at an advanced age.Thus, the medical uses of the present invention are particularlyapplicable for the above-described patient group, for example at the ageof 60, 65, 70, 75, 80 or older and in principle concern all diseasesmanifesting themselves in form of derailment of any kind, like forexample endoproteolysis, conformation alterations, alterations of thepost-translational modifications, somatic mutations, or combinationsthereof, phenotypically by means of developing pathophysiologicalvariants of endogenous protein. In the sense of the present invention,pathophysiological variants are considered to be variants containingpathological neoepitopes that deviate from the physiology, see supra.

In particular, the therapeutic applications include tumor diseases,inflammatory diseases and diseases of the central nervous system, likeAlzheimer's disease, Parkinson's disease, Pick's disease, Dementia withLewy Bodies, Prion diseases including Creutzfeldt-Jakob disease,progressive supranuclear palsy, multiple system atrophy, corticobasaldegeneration, frontotemporal degeneration with Parkinsonism liked tochromosome 17 Huntington's disease, frontotemporal dementia, cerebralamyloid angiopathy, mild cognitive impairment, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis Dutch type and Icelandictype, spinocerebellar ataxia and amyotrohic lateral sclerosis as well asglaucoma, inclusion body myositis, familial amyloid polyneuropathy andamyloidoses comprising fibrillary proteins derived from at least one ofthe following precursor proteins SAA (Serum-Amyloid-Protein A), AL (k or1-light chains of Immunoglobulins), AH (g1 Ig-heavy chains), ATTR(Transthyretin, Serum-Prealbumin), AApo-A-1 (Apolipoprotein A1), AApoA2(Apolipoprotein A2), AGel (Gelsolin), ACys (Cystatin C), ALys(Lysozyme), AFib (Fibrinogen), Beta-amyloid (Amyloid precursor protein),Beta-amyloid2M (beta2-microglobulin), APrP (Prion protein), ACal(Procalcitonin), AIAPP (islet amyloid polypeptide); APro (Prolactin),AIns (Insulin); AMed (Lactadherin); Aker (Kerato-epithelin); ALac(Lactoferrin), Abri (AbriPP), ADan (ADanPP); or AANP (Atrialnatriuretical peptide), (Skovronsky at al., Annu. Rev. Pathol. Mech.Dis. 2006; 1:151-70; Buxbaum, Curr Opin Rheumatol 2003; 16: 67-75.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that antibodies derived from B cells or Bmemory cells from a healthy preclinical or clinically unusually stableorganism are, with a certain probability, capable of preventing aclinically manifest disease, or of diminishing the risk of theoccurrence of a clinically manifest disease, or of delaying the momentof the occurrence of a clinically manifest disease. Typically, suchantibodies also have already successfully gone through somaticmaturation, i.e. the optimization with respect to selectivity andeffectiveness in the high affinity binding to the target molecule bymeans of somatic variation of the variable regions of the antibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical development of theprophylactic or therapeutic antibody in at least one human subject. Itcan thus be expected that, with a procedure according to the presentinvention, both the target structure-specific efficiency of an antibodyas therapeutic agent and its decreased probability of side effectssignificantly increase its clinical probability of success.

From the foregoing, it is evident that the present invention encompassesany use of a disease specific binding molecule comprising at least oneCDR of the above described antibody, in particular for diagnosing and/ortreatment of a disorder related to Alzheimer's disease and Abetadeposition, respectively. Preferably, said binding molecule is anantibody of the present invention or an immunoglobulin chain thereof. Inaddition, the present invention relates to anti-idiotypic antibodies ofany one of the mentioned antibodies described hereinbefore. These areantibodies or other binding molecules which bind to the unique antigenicpeptide sequence located on an antibody's variable region near theantigen binding site.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described binding molecules,antibodies, antigen-binding fragments, polynucleotides, vectors or cellsof the invention and optionally suitable means for detection such asreagents conventionally used in immuno or nucleic acid based diagnosticmethods. The antibodies of the invention are, for example, suited foruse in immunoassays in which they can be utilized in liquid phase orbound to a solid phase carrier. Examples of immunoassays which canutilize the antibody of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay), flow cytometry and the Western blotassay. The antigens and antibodies of the invention can be bound to manydifferent carriers and used to isolate cells specifically bound thereto.Examples of well known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble orinsoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds; seealso the embodiments discussed hereinabove.

By a further embodiment, the binding molecules, in particular antibodiesof the present invention may also be used in a method for the diagnosisof a disorder in an individual by obtaining a body fluid sample from thetested individual which may be a blood sample, a lymph sample or anyother body fluid sample and contacting the body fluid sample with anantibody of the instant invention under conditions enabling theformation of antibody-antigen complexes. The level of such complexes isthen determined by methods known in the art, a level significantlyhigher than that formed in a control sample indicating the disease inthe tested individual. In the same manner, the specific antigen bound bythe antibodies of the invention may also be used. Thus, the presentinvention relates to an in vitro immunoassay comprising the bindingmolecule, e.g., antibody or antigen binding fragment thereof of theinvention.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, a protein- orantibody-based array may be used, which is for example loaded witheither antigens derived from the mentioned disorder-associated proteinand containing the neoepitope in order to detect autoantibodies whichmay be present in patients suffering from, e.g., a neurologicaldisorder, in particular Alzheimer's disease, or with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize any one of those proteins. For example, antigenmicroarray profiling of autoantibodies in rheumatoid arthritis has beenreported by Hueber et al., Arthritis Rheum. 52 (2005), 2645-2655. Designof microarray immunoassays is summarized in Kusnezow et al., Mol. Cell.Proteomics 5 (2006), 1681-1696. Accordingly, the present invention alsorelates to microarrays loaded with binding molecules or antigensidentified in accordance with the present invention.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. binding molecule,antibody or binding fragment thereof, antigen, polynucleotide, vector orcell of the present invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition or alternatively the kit comprisesreagents and/or instructions for use in appropriate diagnostic assays.The composition, e.g. kit of the present invention is of courseparticularly suitable for the diagnosis, prevention and treatment of adisorder which is accompanied with the presence of a disorder-associatedprotein as defined above, especially amyloidosis, and in particularapplicable for the treatment of Alzheimer's disease (AD).

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. The term “treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, e.g. arresting itsdevelopment; or (c) relieving the disease, e.g. causing regression ofthe disease.

Furthermore, the term “subject” or “patient” refers to a mammal,preferably a human, in need of treatment for a condition, disorder ordisease.

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical orintradermal administration. Aerosol formulations such as nasal sprayformulations include purified aqueous or other solutions of the activeagent with preservative agents and isotonic agents. Such formulationsare preferably adjusted to a pH and isotonic state compatible with thenasal mucous membranes. Formulations for rectal or vaginalad-ministration may be presented as a suppository with a suitablecarrier.

Furthermore, whereas the present invention includes the now standard(though fortunately infrequent) procedure of drilling a small hole inthe skull to administer a drug of the present invention, in a preferredaspect, the binding molecule, especially antibody or antibody based drugof the present invention can cross the blood-brain barrier, which allowsfor intravenous or oral administration.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as dopamine orpsychopharmacologic drugs, depending on the intended use of thepharmaceutical composition. Furthermore, the pharmaceutical compositionmay also be formulated as a vaccine, for example, if the pharmaceuticalcomposition of the invention comprises an anti-Aβ antibody for passiveimmunization.

In addition, co-administration or sequential administration of otheragents may be desirable. A therapeutically effective dose or amountrefers to that amount of the active ingredient sufficient to amelioratethe symptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, LD₅₀/ED₅₀. Preferably, the therapeutic agent in the compositionis present in an amount sufficient to restore normal behavior and/orcognitive properties in case of Alzheimer's disease.

The pharmaceutical compositions in accordance with the present inventioncan preferably be used for the treatment of neurological disordersincluding but not limited to Alzheimer's disease, Parkinson's disease,Pick's disease, Dementia with Lewy Bodies, Prion diseases includingCreutzfeldt-Jakob disease, progressive supranuclear palsy, multiplesystem atrophy, corticobasal degeneration, frontotemporal degenerationwith Parkinsonism liked to chromosome 17 Huntington's disease,frontotemporal dementia, cerebral amyloid angiopathy, mild cognitiveimpairment, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis Dutch type and Icelandic type, spinocerebellar ataxia,amyotrohic lateral sclerosis, Bell's Palsy, epilepsy, encephalitis,neuromuscular disorders, glaucoma, inclusion body myositis, familialamyloid polyneuropathy, amyloidoses comprising fibrillary proteinsderived from at least one of the following precursor proteins SAA(Serum-Amyloid-Protein A), AL (k or l-light chains of Immunoglobulins),AH (g1 Ig-heavy chains), ATTR (Transthyretin, Serum-Prealbumin),AApo-A-1 (Apolipoprotein A1), AApoA2 (Apolipoprotein A2), AGel(Gelsolin), ACys (Cystatin C), ALys (Lysozyme), AFib (Fibrinogen),Beta-amyloid (Amyloid precursor protein), Beta-amyloid2M(beta2-microglobulin), APrP (Prion protein), ACal (Procalcitonin), AIAPP(islet amyloid polypeptide); APro (Prolactin), AIns (Insulin); AMed(Lactadherin); Aker (Kerato-epithelin); ALac (Lactoferrin), Abri(AbriPP), ADan (ADanPP); or AANP (Atrial natriuretical peptide),(Skovronsky at al., Annu. Rev. Pathol. Mech. Dis. 2006; 1:151-70;Buxbaum, Curr Opin Rheumatol 2003; 16: 67-75, neuro-oncology,neuro-immunology, neuro-otology pain, pediatric neurology, phobia,affective disorders, sleep disorders, Tourette Syndrome, other movementdisorders and disease of the central nervous system (CNS) in general.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way.Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature; see also “The Merck Manualof Diagnosis and Therapy” Seventeenth Ed. ed by Beers and Berkow (Merck& Co., Inc. 2003).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. For furtherelaboration of general techniques useful in the practice of thisinvention, the practitioner can refer to standard textbooks and reviewsin cell biology and tissue culture; see also the references cited in theexamples. General methods in molecular and cellular biochemistry can befound in such standard textbooks as Molecular Cloning: A LaboratoryManual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); ShortProtocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley& Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985);Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization(Hames and Higgins eds. 1984); Transcription And Translation (Hames andHiggins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss,Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller andCalos, eds.); Current Protocols in Molecular Biology and Short Protocolsin Molecular Biology, 3rd Edition (Ausubel et al., eds.); andRecombinant DNA Methodology (Wu, ed., Academic Press). Gene TransferVectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold SpringHarbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.,eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir andBlackwell, eds., 1986). Protein Methods (Bollag et al., John Wiley &Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. eds.,Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., AcademicPress 1995); Immunology Methods Manual (Lefkovits ed., Academic Press1997); and Cell and Tissue Culture: Laboratory Procedures inBiotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents,cloning vectors and kits for genetic manipulation referred to in thisdisclosure are available from commercial vendors such as BioRad,Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.General techniquesin cell culture and media collection are outlined in Large ScaleMammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997),148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); LargeScale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375);and Suspension Culture of Mammalian Cells (Birch et al., BioprocessTechnol. 19 (1990), 251); Extracting information from cDNA arrays,Herzel et al., CHAOS 11 (2001), 98-107.

The following experiments are illustrated and described with respect toantibody NI-101.11. However, the other antibodies of the NI 101 series,in particular NI 101.10 are structurally similar and thus may beexpected to provide comparable results.

Supplementary Methods Memory B Cell Display

Clinically carefully selected human subjects who are characterized byunusually positive clinical courses, e.g. absence of clinical signs ofdisease in the presence of risk factors, or stable courses of mild orprodromal signs with no disease development, or long-termnon-progressors are recruited to provide peripheral blood lymphocytes asthe starting material for the isolation of memory B cells. The strategyis based upon the concept established in infection immunology that asubject's memory B cell pool preserves the antibody specificities andpossibly also antibody frequencies generated during previous antigenencounters (McHeyzer-Williams and Ahmed Curr. Opin. Immunol. 11 (1999),172.179; Bernasconi et al., Science 298 (2002), 2199-202; Traggiai etal., Nat. Med. 10 (2004), 871-875). This concept was developed toexplain adaptive immunity against infectious agents, as well as for thedescription of antibody-mediated immunity following a primary infection.According to this theory, the entire complement of antibodies againstall antigens that had had induced an antibody response in the subject'shistory, either naturally or upon vaccination, should be fullyrepresented within the memory B cell pool. In accordance with thepresent invention this theory is applied to endogenous antigensgenerated as a result of abnormal aggregation or conformation of anotherwise physiological relevant protein, and that, as such, is notsubject to the physiological immunologic tolerance, and, thus, canacquire antigenic properties and induce an immune response against theconformational neo-epitopes (neoepitopes).

Memory B cells are isolated with surface markers including the pan Bcell marker CD22, combined with negative selection ofantigen-inexperienced B cells that expressed IgM, IgD, IgE, and IgA.With this technique, approximately 10.000 to 150.000 memory B cells canbe obtained from 30 ml of human blood. These are immortalized, forexample with Epstein Barr Virus, and cultured oligo-clonally onirradiated human fibroblast feeder layers (Zubler et al., J. Immunol.134 (1985), 3662-3668; Traggiai et al., Nat. Med. 10 (2004), 871-875).To improve transformation and immortalization efficacy ofantibody-secreting memory B cells, CpG 2006 which mimics the activitiesof bacterial un-methylated CpG-dinucleotides (Hartmann and Krieg JImmunol 164(2) (2000), 944-953) can be used.

Experimental Protocol:

Selection of B cells from the bulk of PBL was performed using the MACStechnology and CD22 microbeads (Miltenyi, Bergisch Gladbach, Germany).PBL were labeled with MACS anti human CD22, phycoerythrin-conjugatedanti human IgD and APC-conjugated antibodies anti human IgM, IgA, CD3,CD8, CD56 (Becton Dickinson, Basel, Switzerland). CD22-positive cellswere isolated using LS columns and the Midi MACS device (Miltenyi)followed by selection of phycoerythrin- and APC-negative cells using aMoFlo cell sorter (Dako, Fort Collins, USA), CD22-positive, IgM-, IgD-,IgA-, and IgE-negative B cells were then incubated with Epstein BarrVirus containing supernatant obtained from B95-8 cells and CpG 2006(Sigma, Buchs, Switzerland) at a concentration of 2.5 mg/l in B cellmedium (RPMI 1640 supplemented with 10% fetal calf serum (Hyclone,Perbio, Lausanne, Switzerland). 5-50 cells were seeded per well inCostar round bottom 96 well plates (Corning, Vitaris, Baar, Switzerland)in B cell medium on 30.000 irradiated human PBL prepared from voluntarydonors. Memory B cell cultures were maintained at 37° C. and 5% CO₂ in ahumidified cell culture incubator for 2-4 weeks after which time theconditioned medium of the cultures was assayed in ELISA and on tissuearrays.

Antibody Screening

Antibodies in conditioned media are screened for binding to pathologicalepitopes including protein aggregates and abnormal, pathologicallyrelevant structures on tissue sections obtained from human patients withpathologically confirmed diagnoses including, but not restricted to,Alzheimer's disease, or obtained from tissue sections of transgenicmouse models of human disease, or from tissue sections obtained fromanimal models of human disease including aged non-human primates, or byELISA of aggregated synthetic peptide preparations. Abnormal, pathologicstructures in the sense of this invention include, but are notrestricted to, β-amyloid plaques, neurofibrillary tangles,alpha-synuclein aggregates in Lewy bodies, and protein aggregatesdeposited in dystrophic neurites. Human tissues are also used to excludecross-reactivities of antibodies with normal cellular or supercellulartissue structures. Selected antibodies are further analyzed for classand light chain subclass determination. Selected pathology-relevantantibody messages from memory B cell cultures are transcribed by usingRT-PCR, cloned and combined into expression vectors for recombinantproduction.

Experimental Protocol:

Screening of B cell conditioned medium using microliter-compatibletissue microarrays.

Array Production

Paraffin-embedded human post-mortem Alzheimer's disease brain tissueswere cut into rods of 1-2 mm in diameter and 10 mm in length. Four rodswere embedded vertically in paraffin to form a square fitting themicrotiter format of 9 by 9 mm. 5 μm tissue slices were cut from thisassembly with a microtome and two slices were mounted adjacent to eachother onto glass slides resulting in an assembly of 2 by 4 rods that fitthe 96-well microtiter format. Alternatively, tissues from APPtransgenic mice were used to prepare the tissue arrays.

B Cell Screening

Conditioned medium from memory B cell cultures was transferred onto thetissue array slides using a multichannel pipette and incubated for 2 hat room temperature. After a washing step the binding of humanantibodies to tissue sections was analyzed using Cy 3-conjugatedsecondary antibodies to human IgG (Jackson ImmunoResearch Europe Ltd.,Suffolk, UK). Analysis of fluorescence was performed on an invertedfluorescence microscope (Leica, Heerbrugg, Switzerland).

ELISA

96 well half area Microplates (Corning) were coated with syntheticAbeta-peptide at a standard concentration of 1 μg/ml in coating buffer(15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.42) overnight at 4° C. Plates werewashed and non-specific binding sites were blocked for 1 h at RT withPBS containing 2% BSA (Sigma, Buchs, Switzerland). B cell conditionedmedium was transferred from memory B cell culture plates to ELISA platesand was incubated for 2 h at room temperature. Binding of humanantibodies was determined using horse radish peroxidase (HRP)-conjugateddonkey anti-human IgG polyclonal antibodies (Jackson ImmunoResearchEurope Ltd., Cambridgeshire, UK) followed by measurement of HRP activityin a standard colorimetric assay.

Molecular Cloning of Antibodies Displaying Specificity of Interest

Living B cells of selected memory B cell cultures are harvested using acell sorter.

mRNA is prepared and immunoglobulin heavy and light chain sequences areobtained using Ig-framework specific primers for all human variableheavy and light chain framework 1 (FR1) families as 5′ primers incombination with primers specific for all human J-H segments as 3′primers (Marks et al., Mol. Biol. 222 (1991)., 581-597). Alternatively,single-cell RT-PCR of single sorted cells from the memory B cell culturecan be used as source of Ig heavy and light chain sequences (Babcook etal., Proc. Natl. Acad. Sci. USA 93 (1996), 7843-7848; Brezinschek etal., J. Immunol. 155 (1995), 190-202; Coronella et al., Nucleic AcidsResearch 28 (2000); Owens, et al., J. Immunol. 171 (2003), 2725-2733).Single cell sorting preserves the correct pairing of the immunoglobulinheavy and light chains of the antibody clones originally produced in theB cell culture.

Identification of the antibody clone with the desired specificity isperformed by re-screening on microtiter compatible tissue microarray andELISA upon recombinant expression of complete antibodies. Recombinantexpression of complete IgG1 antibodies is achieved upon insertion of thevariable heavy and light chain sequences “in the correct reading frame”into expression vectors that complement the variable region sequencewith a sequence encoding a signal peptide at the 5-prime end and at the3′-end with a sequence encoding the appropriate constant domain(s). Tothat end the primers contained restriction sites designed to facilitatecloning of the variable heavy and light chain sequences into antibodyexpression vectors. Heavy chain immunoglobulin are expressed byinserting the immunoglobulin heavy chain RT-PCR product in frame into aheavy chain expression vector bearing a signal peptide and the constantdomains of human immunoglobulin. Kappa light chain immunoglobulin isexpressed by inserting the kappa light chain RT-PCR-product in frameinto a light chain expression vector providing a signal peptide and theconstant domain 1 of human kappa light chain immunoglobulin.Alternatively, lambda light chain immunoglobulin is expressed byinserting the lambda light chain RT-PCR-product in frame into a lambdalight chain expression vector providing a signal peptide and theconstant domain 1 of human lambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies are obtained uponco-transfection into HEK 293 cells (or any other appropriate recipientcell line) of a Ig-heavy-chain expression vector and a kappa or lambdaIg-light-chain expression vector. Recombinant human monoclonal antibodyis subsequently purified from the conditioned medium using a standardProtein A column purification. Recombinant human monoclonal antibody canbe produced in unlimited quantities using either transiently or stablytransfected cells. Cell lines producing recombinant human monoclonalantibody can be established either by using the Ig-expression vectorsdirectly or by re-cloning of Ig-variable regions into differentexpression vectors. Derivatives such as F(ab), F(ab)₂ and scFv can alsobe generated from these Ig-variable regions.

Experimental Protocol: RT-PCR of Bulk B Cells

Living cells as identified by their forward- and sideward lightscattering properties of selected memory B cell cultures were sorted inaliquots of 100-2000 cells directly in 0.2 ml PCR tubes filled with 20μl of RNA later (Ambion, Huntingdon, UK) using a MoFlo cell sorter. mRNAwas prepared using the mRNA-Direct Micro Kit (Dynal, Invitrogen, Basel,Switzerland). cDNA was prepared using the “RT for PCR” Kit (ClontechBectonDickinson, Basel, Switzerland) and PCR of immunoglobulin (Ig)heavy and light chain variable sequences was performed using theAdvantage 2 PCR Kit (Clontech) using specific primers for all humanvariable heavy and light chain frame work 1 (FR1) families as 5′ primersin combination with primers specific for the constant domains of humanIg heavy or Ig kappa or Ig lambda light chains as 3′ primers. Primerswere purchased from Microsynth (Balgach, Switzerland).

A signal peptide that was used in all expression vectors was derivedfrom the human immunoglobulin kappa light chain family 1 L5 sequence(MDMRVPAQLLGLLLLWFPGSRC SEQ ID NO: 2) as described at V-Base anddesigned to provide the restriction site Xba 1 in order to facilitatethe cloning of PCR amplified variable regions

(ATGGACATGCGGGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCTAGATGC;SEQ ID NO: 1). Xba1 was introduced by silent mutagenesis. As a 3′restriction site used for the cloning of variable heavy chain regionsthe restriction site Sal1 was introduced into C1 of IgG1 provided by thevector. Similarly, the restriction site BsiW1 was introduced into C1 ofthe kappa light chain and Xho1 was introduced into C1 of lambda lightchain. Restriction digest of PCR products and ligation into to recipientvectors was performed according to standard procedures. Plasmid DNA wasprepared using standard kits (Quiagen, Hombrechtikon, Switzerland).Recipient vectors contained a CMV promoter for the expression ofantibody genes in mammalian cells.

Single-Cell RT-PCR

For single cell RT-PCR of Ig heavy and light chain variable regions fromcultured B cells a modification of the method described by Owens et al.was used (Owens et al., 2003). Single cells were deposited directly intoeach tube of an array of 0.2 ml PCR tubes using the MoFlo cell sorter.Each tube was prepared to contain 10 μl of RT buffer for Superscript IIreverse transcriptase (Invitrogen). PCR tubes were shock frozen on dryice and thawed immediately prior to RT-PCR. cDNA was prepared usingrandom hexamers (Clontech) and Superscript II reverse transcriptase(Invitrogen). 1^(st) round PCR of immunoglobulin heavy and light chainvariable regions was performed using, as 5′ primers, a set of primersthat primed in all signal peptides conserved among Ig-variable regionfamilies. At the 3′ position, single primers specific for the constantregion of Ig C1 heavy chain or the Ig kappa- or Ig lambda light chainwere used. 2nd round PCR was performed on PCR-products obtained duringfirst round PCR using the primers as described for the bulk B cellRT-PCR. Cloning into recipient vectors was performed accordingly.

Alternative Cellular Cloning

Cloning was performed using the standard limited dilution method or bysingle cell deposition into 96 well culture plates using a cell sorter(MoFlo, Dako, Fort Collins, USA). For limiting dilution, cells of amemory B cell culture were harvested, passed through a 30 μm nylon mesh(Falcon, Becton Dickinson, Basel, Switzerland) resuspended in medium andseeded onto 96 well plates or 384 well plates at a concentration of 0.3cells per well.

For seeding with the cell sorter, the device was set to deposit onesingle cell (single 1 mode) per well directly in 96 well platessupplemented with B cell medium. The culture medium was supplementedwith medium conditioned by activated T cells. Alternatively, 30.000irradiated feeder cells were added to the medium.

Sequence Analysis of Immunoglobulin Variable Region Sequences

Sequencing of cloned immunoglobulin variable region sequences wasperformed using primers specific for the CMV promoter present 5′ of theinserted Ig variable region sequences in the expression vector.Alternatively, primers that primed in the constant domains of Ig heavy-and light chains were used. Sequences obtained were analyzed and alignedusing Vector NTI software (Informax-Invitrogen). Plasmids containingsequences that encoded complete immunoglobulin variable regions in framewith the leader peptide and the constant domain were used forexpression.

Expression of Functional Recombinant Monoclonal Antibodies

The antibodies can be produced in sufficient quantities by recombinantexpression using technologies known in the art (Trill et al., Curr.Opin. Biotechnol. 6 (1995), 553-601). Recombinant human monoclonalantibody of up to 1 mg was produced upon transient transfection of 293HEK cells. Recombinant human monoclonal antibody of up to 100 mg wasproduced upon stable transduction of 293 HEK cells or the murine NSOcells using recombinant lentivirus vectors.

Small Scale Production of Human Recombinant Antibody by TransientTransfection

Ig-heavy chain vector and Ig-light chain vectors were co-transfectedinto HEK 293 cells using the standard calcium phosphate co-precipitationmethod. Recombinant antibodies were purified from the medium conditionedby transfected HEK 293 cells using protein A column purification(GE-Healthcare, Otelfingen, Switzerland).

Large Scale Production of Human Recombinant Antibody by StableTransduction

Here, a lentivirus-based transfection system was employed to generatestably transduced cell lines producing human recombinant antibody(Zufferey et al., J. Virol. 72 (1998), 9873-9880). HEK 293 cells wereco-transduced with two distinct lentivectors one bearing an expressioncassette for the Ig heavy chain, the other a cassette for the Ig lightchain of a recombinant antibody. This method of transduction can be usedin a broad range of mammalian cell lines such as CHO and NSO cells.

Validation in Transgenic Mouse Models of Human Disease

Transgenic mice were generated as previously described (Knobloch et al.,Neurobiol Aging Jul. 28 (2006)) on a hybrid background of C57B1/6 andDBA2. The test group was backcrossed once to C57B1/6. Mice were keptunder standard housing conditions on a reversed 12 h:12 h light/darkcycle and had free access to food and water. The treatment groups werebalanced for age (24 months at first testing, 26 months at 2^(nd)testing) and gender. Mice are treated with antibodies (3 mg/kg bodyweight) by once weekly intraperitoneal injections over a time period of2 months resulting in 8 injections per animal.

Behavioral Testing in the Y-Maze

The spontaneous alternation rate is assessed using a Y-shaped plasticmaze, with 40×20×10 cm arm sizes. During 5 min sessions, the sequencesof arm entries are recorded; alternation was defined as successiveentries into the three arms, in overlapping triplet sets. The percentalternation was calculated as the ratio of actual to possiblealternations. After 2 months of antibody treatment, the mice areretested in the Y-maze. The experimenters are always kept blind for bothtreatments and genotypes during the whole experiment.

Blood-Brain Barrier Penetration and Binding to Abnormal Structures inthe Brain

To assess whether the selected antibodies or fragments thereof canpenetrate the blood-brain barrier and bind to their abnormallyaggregated or conformationally altered protein targets in the brain, aneffective dose of the antibody is administered systemically,intraperitoneally, intravenously, intramuscularly, subcutaneously orintranasally to a transgenic animal that is characterized byunphysiological accumulation of the aggregated or conformationallyaltered protein target in the brain. Binding of the antibody to thepathology specific structures in the brain is then evaluated byimmunostaining with a labeled anti-human Ig secondary antibody followedby standard immunohistochemical detection.

Experimental Protocol:

PS-1/APPswe transgenic model mice for Alzheimer's disease received twoperipheral injections of 150 μg NI-101.11 at day 1 and day 3. The micewere sacrificed 24 h after the second injection and perfused with PBS.Brains were frozen and tissue slices were prepared from frozen tissueusing a cryotome. Presence of human antibody on cryostat sections wasassayed by staining with Cy3-labeled anti human IgG antibody (JacksonImmunoResearch Europe, Suffolk, UK). Localization of amyloid plaques wasperformed by co-staining the cryostat sections with the murineAbeta-specific control antibody 6E10 (available from Covance, CatalogNumber SIG-39320) followed by FITC-labeled anti mouse IgG antibody.Alternatively, staining with Cy3-labeled anti human IgG antibody wasused alone. Analysis of fluorescence was performed on an invertedfluorescence microscope (Leica).

Reduction of Brain Pathology

The effects of antibody treatment on the levels of aggregated orconformationally altered protein targets in the brain is assessed bysystemic treatment or targeted brain delivery of the antibody(intracranial, intrathecal or intraventricular) and an unrelatedantibody control to transgenic animals with characteristicunphysiological accumulation of the aggregated or conformationallyaltered protein target in the brain. The treatment effects is evaluatedby immunostaining or histochemical staining of the altered or aggregatedprotein targets and measuring the area covered by such aggregates,aggregate size and aggregate number and biochemical quantification ofthe concentrations of the protein targets in different brain areas.

Absence of Antibody-Treatment Related Side Effects

Potential target related adverse effects of the antibody-treatment willbe assessed by systemic administration or targeted brain delivery of theantibody (intracrainial, intrathecal or intraventricular) and anunrelated antibody control to transgenic animals with characteristicunphysiological accumulation of the aggregated or conformationallyaltered protein target in the brain. Potential side effects will beevaluated by immunostaining or histochemical staining (e.g. Prussianblue for micorhemorrhages, hematoxilin-eosin, activated white bloodcells) and biochemical quantification (e.g. cytokine levels by ELISA).

Immunofluorescence Staining of Living Cells

HEK 293 cells were transiently transfected with a vector that expresseshuman wild type APP fused at the intracellular C-terminus to the yellowfluorescent protein variant Citrine. 24 hours after transfection thecells were incubated with human recombinant antibodies or controlantibodies at 4° C. for 30 minutes. After a washing step the cell werefixed and surface-bound antibody was detected using Cy-3-labeledsecondary antibodies to human or mouse IgG (Jackson ImmunoResearch).Analysis of fluorescence was performed on a confocal microscope (Leica).

Preparation of Abeta Fibrils

Abeta peptide was purchased from Bachem (Bubendorf, Switzerland).Lyophylised peptide was reconstituted in TFA and resuspended in PBSimmediately prior to its use as monomeric Abeta in the assays. Abetafibrils were prepared by incubation of monomeric Abeta1-42 peptide at aconcentration of 100 μg/ml in PBS at 37° C. for 24 h. Monomeric Abetapeptide and fibril preparations were also used as substrate to coatELISA plates.

Western Blotting

Monomeric Abeta peptide was mixed with loading dye, heat denatured and0.2 μg was loaded per lane and separated on a gradient SDS-PAGE. Blotswere incubated with primary antibody for 2 h. Binding of primary humanmonoclonal antibody or mouse control antibody 6E10 was revealed usingsecondary anti human or anti mouse antibodies conjugated with horseradish peroxidase (HRP). Blots were developed using SuperSignal WestFemto Maximum Sensitivity Substrate (Pierce, Fisher Scientific, Wohlen,Switzerland).

Competition of Tissue Amyloid Plaque Binding

Recombinant human NI-101.11 antibody was incubated for 2 h with Abetapeptide preparations. The antibody/Abeta preparations were then used forimmunohistochemical staining of brain section obtained from a patientwith neuropathologically confirmed Alzheimer's disease. 5 μmcryo-sections were prepared, blocked with 4% BSA, 5% goat serum and 5%horse serum in PBS for 1 h at RT and stained with NI-101.11/Abetapreparations for 1 h at room temperature. After a washing step thebinding of human antibodies to tissue sections was analyzed usingCy3-conjugated secondary antibodies to human IgG (Jackson ImmunoResearchEurope Ltd). Analysis of fluorescence was performed on an invertedfluorescence microscope (Leica, Heerbrugg, Switzerland).

Example 1 Detection of Human Antibodies Against Abnormal StructuresPrevalent in Human Brain Diseases

Antibodies from phenotypically healthy subjects, or clinically unusuallystable patients with Alzheimer's disease were tested byimmunohistochemistry on brain sections obtained from patients withpathologically confirmed Alzheimer's disease. FIG. 1A demonstrates thepresence of antibodies in a clinically unusually stable patient thatbind to beta-amyloid plaques as was confirmed by co-staining with aknown antibody against human beta-amyloid (antibody 4G8; FIG. 1B). Thepresence in a healthy human subject of antibodies to neurofibrillarytangles in a tissue section obtained from a patient with Alzheimer'sdisease is shown in FIG. 2A. This result was confirmed by co-stainingwith a known antibody against human tau (HT7). FIG. 3A reveals thepresence in a healthy human subject of antibodies against dystrophicneurites in a tissue section obtained from a patient with Alzheimer'sdisease. Control staining with known antibody against human tau (HT7) isdepicted in FIG. 3 B. These results demonstrate the presence inphenotypically healthy, or clinically unusually stable patients ofantibodies against identifiable pathological structures in human tissuesamples with histopathologically confirmed diagnoses.

Example 2 Recombinant Human Antibodies Maintain Specificity to AbnormalStructure In Vivo and Recognize Conformational Epitope ofDisease-Associated Beta-Amyloid Protein in Brain Amyloid Plaques but notthe Physiological Precursor or Non-Pathogenic Derivative Thereof

Antibodies NI-101.11, NI-101.12, NI-101.13A and NI-101.13B were obtainedfrom clinically unusually stable Alzheimer's disease patients with asignificantly reduced rate of cognitive decline. Antibody isolation andrecombinant production was performed as specified in supplementarymethods.

Recombinant NI-101.11 was tested for binding to brain beta-amyloidplaques (FIG. 4). Brain sections obtained from a patient withneuropathologically confirmed Alzheimer's disease were stained at theindicated concentrations. Antibody binding to beta-amyloid plaques withconcentrations of 50 pM suggest high affinity binding. The binding ofantibody NI-101.11 to beta-amyloid plaques at a concentration of 0.5 nMcannot be competed by addition of excess amounts of linear syntheticN-terminal Abeta-derived polypeptide representing positions 1 to 16 atconcentrations of up to 1 μM (FIG. 5). Furthermore, the binding ofNI-101.11 to beta-amyloid plaques on brain sections at 8 nMconcentration is competed by excess amounts Abeta1-42 fibrils (4 μM) butnot of linear synthetic Abeta1-42 monomers at 4 μM concentration,suggesting that NI-101.11 recognizes a conformational epitope that isnot present in monomeric Abeta (FIG. 6).

To further assess binding of human recombinant NI-101.11 antibody tolinear, monomeric synthetic Abeta, preparations of monomeric Abeta wereseparated by non-denaturing PAGE. Blotted protein was probed with humanrecombinant NI-101.11 antibody and a control antibody against N-terminallinear Abeta sequences (6E10). While 6E10 produced prominent staining ofmonomeric Abeta peptide, no binding was detected for human NI-101.11suggesting that NI-101.11 does not bind to the linear monomeric Abetapeptide but recognizes a conformational Abeta epitope. (FIG. 7)

The binding of recombinant NI-101.11 to artificial amyloid fibrilsprepared from synthetic Abeta1-42 peptides and monomeric Abeta wasdetermined by ELISA (FIG. 8). Synthetic Abeta fibrils or monomericsynthetic Abeta coated onto ELISA plates at equal coating densities wereincubated with NI-101.11 at the indicated concentrations. Binding toartificial amyloid fibrils (open squares) is more than 100 times higheras compared to monomeric Abeta (filled squares). Control antibody 22C4against the C-terminus of Abeta preferentially binds to monomeric Abeta(filled circles), and less well to fibrils (open circles). This suggeststhat NI-101-10 recognizes a conformational epitope which is also presenton artificial amyloid fibrils prepared from synthetic Abeta peptides.

Cross-reactivity of recombinant human NI-101.11 antibody againstcellular full-length APP or with any of its physiological derivativeswas determined by cell binding assays (FIG. 9).

Live HEK 293 cells stably expressing human APP fused to Citrin as amarker were incubated for 30 min at 4° C., to prevent internalization,with the recombinant human NI-101.11 antibody or the control antibody6E10 against N-terminal linear Abeta sequence. Citrin-positive signalsindicate APP-expressing cells. In contrast to the control antibody(6E10) that binds to cell-surface APP in all cells expressing the fusionconstruct, no binding of recombinant human NI-101.11 antibody tofull-length APP is detected. These data demonstrate absentcross-reactivity of NI-101.11 to physiological, cellular APP.

The lack of binding of NI-101.11 to monomeric Abeta was furtherdemonstrated by size exclusion chromatography: No binding of NI-101.11or an unrelated control antibody was observed to monomeric FITC-labeledAbeta1-42 (FIGS. 10A, 10B). In contrast, antibody 22C4 directed againsta linear epitope present in the C-terminus of Abeta co-eluted withFITC-Abeta1-42 monomers (FIG. 10 C).

In a competition ELISA, binding of 6E10, an antibody directed against alinear epitope at the N-terminus of Abeta, could be completely blockedupon pre-incubation with excess concentrations of monomeric Abeta 1-16,Abeta 1-28 and Abeta 1-40 peptides. In contrast, pre-incubation withexcess concentrations of linear Abeta peptides did not abolish NI-101.11binding, suggesting that NI-101.11 requires a conformational epitope(FIG. 11).

NI-101.13A and 13B

Recombinant human antibodies NI-101.13A and 13B were tested for bindingto brain sections obtained from an APP transgenic mouse model ofAlzheimer's disease (Tg2576). NI-101.13A and NI-101.13B producedprominent staining of beta-amyloid plaques at 10 nM concentration (FIG.12). The binding of recombinant NI-101.13A and NI-101.13B to artificialamyloid fibrils prepared from synthetic Abeta1-42 peptides and monomericAbeta was determined by ELISA. Synthetic Abeta fibrils or monomericsynthetic Abeta coated onto ELISA plates at equal coating densities wereincubated with NI-101.13A and NI-101.13B at the indicatedconcentrations. Preferential binding to artificial amyloid fibrils ascompared to monomeric Abeta was observed for both antibodies tested.(FIG. 13)

NI-101.12

The binding of recombinant NI-101.12 to synthetic Abeta1-42 peptide wasconfirmed by ELISA (FIG. 14A). NI-101.12 binding at 133 nM concentrationwas competed by excess of Abeta1-42 peptide (FIG. 14 B).

Example 3 Recombinant Human Antibody Against Brain Beta-Amyloid Crossesthe Blood Brain Barrier in a Transgenic Mouse Model of Alzheimer'sDisease, and Binds to Brain Beta-Amyloid Plaques In Vivo

To determine whether recombinant human NI-101.11 antibody crosses theblood brain barrier and binds to brain beta-amyloid plaques in vivotransgenic PS-1/APPswe Alzheimer's disease model mice received twoperipheral injections of 150 μg NI-101.11 at day 1 and day 3. Mice weresacrificed 24 h after the second injection and perfused with PBS. Brainswere harvested and brain sections were stained with FITC-labeledantibodies against human IgG or with the mouse monoclonal Abeta antibody6E10 followed by a FITC-labeled antibody against mouse IgG to confirmthe presence of brain beta-amyloid plaques. Intense staining of amyloidplaques with anti-human IgG indicated that the recombinant humanNI-101.11 antibody can cross the blood-brain barrier of transgenic miceand bind to brain beta-amyloid plaques in living animals (FIG. 15).

Example 4 Recombinant Human Antibody Against Beta-Amyloid ImprovesAbnormal Cognitive Behavior and Confers Reduction of Beta-Amyloid PlaqueLoad, Astrogliosis and Microgliosis in a Transgenic Mouse Model ofAlzheimer's Disease without Increasing the Frequency of Microhemorrhages

24 months old arc Abeta mice and age-matched wild type littermates weretreated weekly i.p. with 3 mg/kg recombinant human NI-101.11 antibody oran isotype-matched human control antibody for 2 months. To assess thetreatment effect on abnormal behavior in the transgenic mice, Y-mazebehavioral testing was performed before and after completion of thetreatment. The spontaneous rate of alternation was assessed using aY-shaped plastic maze, with 40×20×10 cm arm sizes. During 5 minsessions, the sequences of arm entries were recorded; alternation wasdefined as successive entries into the three arms, in overlappingtriplet sets. The percent alternation was calculated as the ratio ofactual to possible alternations (defined as the total number of armentries—2) multiplied by 100%. The Y-maze performance of untreated arcAbeta mice and wildtype littermate controls was compared using anunpaired t-test. The nonparametric Kruskal-Wallis test was used tocompare the improvement after treatment in all 4 groups. Thenonparametric Mann-Whitney U test was chosen for pair-wise comparison ofthe different groups. Zero-performers (i.e. mice that did not leave thearm they were placed in) were excluded from the analysis.

As was observerd in previous studies, untreated 24-months old arc Abetamice were significantly impaired compared to their wildtype littermates(FIG. 16A, before treatment; unpaired t-test, p=0.0007).

NI-101.11 treated arc Abeta mice showed clearly enhanced alterationlevels, comparable to NI-101.11 treated wildtype control mice after the2 months treatment. Analysis of the improvement (i.e. performance aftertreatment minus performance before treatment) showed a significantdifference between the four groups (FIG. 16 B, Kruskal-Wallis test;p=0.03). A pair-wise post-hoc analysis between all groups showed thatNI-101.11 treated arc Abeta mice improved their cognitive performancesignificantly more than wildtype mice (Mann-Whitney U; p=0.05 NI-101.11tg vs. NI-101.11 wt; p=0.008 NI-101.11 tg vs. control wt). This group ofmice also showed a strong trend towards improved performance compared tothe control antibody treated transgenic littermates (Mann-Whitney-U;p=0.08 NI-101.11 tg vs. control tg). All mice showed a ˜10% improvementin performance in the re-testing, which was likely due to the familiarenvironment of the task.

The effects of chronic, 2 months NI-101.11 treatment on amyloid burden,astrogliosis and microgliosis were analyzed by quantitativehistochemical and immunohistochemical analysis. To that end, mice wereanesthetized after completion of the behavioral testing and perfusedtranscardially with PBS. One brain hemisphere was fixed in 4%paraformaldehyde and embedded in paraffin. 5 μm sagittal sections werecut with a Leica RM 2135 microtome (Bannockburn, Ill.). Beta-amyloidplaque load in cortex and hippocampus was quantified on brain sectionsstained with Thioflavin S and Congo Red according to standard protocol.For immunohistochemistry, slices were dewaxed, blocked with 4% BSA, 5%goat serum and 5% horse serum in PBS for 1 h at RT. Antibodies wereincubated overnight at 4° C. using the following dilutions: anti GFAP(Advanced Immunochemicals) 1:500, anti IBA1 (WAKO) 1:500. 2ndfluorophore coupled antibodies were incubated at RT for 2 h. 2-3sections per mouse brain spaced 75 μm apart were used for each staining.2 images per section were taken at 10× magnification for cortex analysis(parietal and frontal region). The entire hippocampus area (5×magnification cropped to ROI) was taken for the hippocampus analysis.Automated image analysis was done with the software ImageJ.

Double staining of brain sections from immunized arc Abeta mice with6E10 and anti-human IgG revealed binding of NI-101.11 to Abeta deposits(FIG. 17, left panel), indicating that NI-101.11 can cross the bloodbrain barrier and bind to brain beta-amyloid plaques. No such binding ofhuman antibody to Abeta deposits was seen in control antibody treatedarc Abeta mice (FIG. 17 right panel).

Chronic treatment with 3 mg/kg of NI-101.11 resulted in a significantreduction of amyloid plaque load as was revealed by Thioflavin S andCongo Red staining. This reduction reached levels of greater than 50% incortex and hippocampus compared to control antibody-treated arc Abetamice (FIGS. 18 A, B). In addition to the plaque area (FIG. 18 C),significant reductions were also observed for the number of plaques(FIG. 18 D) and the average plaque size (FIG. 18 E).

To test whether chronic treatment with NI-101.11 affects theneuroinflammatory response in arc Abeta mice, reactive astrocytes andmicroglia were quantified after immunohistological staining. A reductionin the number of reactive astrocytes (anti GFAP-staining) was observedin the cortex of NI-101.11 treated arc Abeta mice compared to controlantibody treated animals (FIG. 19A; Mann-Whitney-U; p=0.047). No changewas detected in the hippocampus. Staining with an antibody against amarker of microglia and macrophages (anti-Iba1) also revealed astatistical trend towards reduced inflammation (FIG. 19 B;Mann-Whitney-U; p=0.075 for both cortex and hippocampus). The decreasein astrocytosis and microgliosis is in line with the reducedbeta-amyloid load observed after NI-101.11 treatment

Passive immunotherapy with certain monoclonal antibodies directedagainst Abeta can be associated with increased frequency ofmicrohemorrhages in the brain (Pfeifer et al., Science 298 (2002), 1379;Wilcock et al., J Neuroinflammation 1 (2004), 24). To assess the effectsof chronic therapy with NI-101.11, Perl's Prussian blue staining wasperformed on brain sections from arc Abeta and wild-type mice afterchronic NI-101.11 treatment. This staining reveals the presence ofhemosiderin, a breakdown product of hemoglobin, and marker of previousmicrohemorrages (FIG. 20). In aged arc Abeta mice treated with a controlantibody, the frequency of Prussian blue positive profiles wassignificantly elevated compared to wild-type littermates(Mann-Whitney-U; p=0.001). Treatment with the NI-101.11 did not lead toan increase the number of microhemorraghes when compared tocontrol-antibody treated arc Abeta mice (Mann-Whitney-U; p=0.347)indicating that the beneficial therapeutic effects of NI-101.11treatment occurred in the absence of this frequently observed sideeffect of passive Abeta immunotherapy.

Example 5 Recombinant Human Antibody Against Brain Beta-Amyloid Inhibitsthe Formation of Synthetic Abeta Fibrils In Vitro

The effect of recombinant human NI-101.11 antibody on the formation ofAbeta-fibrils was assayed by measuring Thioflavin S bound to aggregatedAbeta by fluorescence analysis. Monomeric Abeta solutions were incubatedat 37° C. for 24 h in the presence of absence of increasingconcentration of NI-101.11. The formation of synthetic Abeta fibrils invitro was inhibited by recombinant human NI-101.11 in a concentrationdependent manner (FIG. 21).

Example 6 NI-101.11 Effects on Ex-Vivo Phagocytosis of Abeta Fibrils byBV-2 Microglial-Derived Cells

The effects of NI-101.11 on Fcgamma-receptor mediated phagocytosis ofAbeta fibrils were studied in the BV-2 microglial-derived cell line.BV-2 cells were maintained in DMEM supplemented with 5% FBS, Pen/Stepand glutamine. Cells were trypsinized and 120'000 BV-2 cells/well wereseeded in flat bottom 24-well plates. After 12 h, the medium wasreplaced with 400 ul DMEM/F 12/well supplemented with 20 mM HEPES (pH7.3), 1% BSA, 10 μg/ml Pen/Step. 100 μg/ml Fucoidan, an inhibitor of thescavenger receptor, was added 30 min prior to experiment. 50 μMFITC-labeled Abeta fibrils were pre-incubated with the indicatedconcentrations of antibodies for 30 min at 37° C., washed twice followedby centrifugation for 5 min at 14'000×g. This suspension was added tothe tissue culture plates. After 30 min BV-2 cells were washed twicewith HBSS to remove unassociated fibrillar Abeta.

Cells were treated with 250 μg/ml trypsin/EDTA for 20 min at 4° C. andwashed twice by centrifugation at 500×g for 5 min at 4° C. Cells werefixed for 20 min in FACS-Fix (PBS, 2% FA, 2% Glucose, 5 mM NaN) andwashed twice with FACS wash (PBS, 5 μM EDTA, 0.2% BSA). Fluorescence(FL-1) of 10'000 cells was determined by FACS analysis (based on WebsterS D et al, JI 2001).

Fcgamma receptor-dependent phagocytosis of FITC-labeled Abeta1-42fibrils was measured upon inhibition of the scavenger receptor system.Comparative analysis of human NI-101.11 and a commercially availableantibody directed to a linear epitope at the N-terminus of the Abetapeptide (6E10) demonstrated dose-dependent induction of phagocytosis ofAbeta fibrils. The uptake of fibrils mediated by NI-101.11 is up to 3fold higher than that observed for the 6E10 antibody (FIG. 22). Thesedata indicate that NI-101.11 triggers potent dose-dependent Fcgammareceptor-mediated phagocytosis of Abeta fibrils by microglial cells.

CONCLUSION

As demonstrated in the above experiments performed in accordance withthe present invention it was surprisingly possible to detect protectiveand therapeutically active antibodies and antibody producing B-cells inphenotypically healthy, asymptomatic human subjects, as well as inpatients with unusually stable clinical disease courses despite adiagnosis of cognitive impairment or Alzheimer's disease. Morespecifically, a new class of human antibodies could be detected andisolated, which discriminate the physiologically functional form of anantigen, thereby minimizing the risk of autoimmunogenic side effectshitherto being a problem in immunotherapy. Thus, antibodies andequivalent binding molecules are provided that specifically recognize avariant of the antigen in a pathophysiologically relevant structure,which the antibody is supposed to bind in order to diminish its toxicityor to reduce its concentration or to promote its degradation, by meansof, for example, making the pathogen for FcR-expressing macrophages ormicroglia cells visible and therefore to render it innocuous. As furtherdemonstrated in the examples such antibodies are therapeuticallyeffective and are capable of both suspending as well as preventingdeleterious effects of abnormal pathological proteins and aggregatesthereof without increasing the frequency of brain microhemorrhages.

1. A recombinantly produced binding molecule, which can selectivelyrecognize a neoepitope of a disorder-associated protein, and which doesnot substantially recognize the protein in its non-disorder-associatedform.
 2. The binding molecule of claim 1, which is an antibody orantigen-binding fragment thereof.
 3. The antibody or antigen-bindingfragment thereof of claim 2, which is a human antibody.
 4. The bindingmolecule of claim 1, which can bind to beta-amyloid plaques,cerebrovascular amyloid, diffuse Abeta deposits, neurofibrillarytangles, hyperphosphorylated tau, alpha-synuclein positive Lewy-bodiesor protein aggregates associated with dystrophic neurites.
 5. Thebinding molecule of claim 1, which is an antibody or antigen-bindingfragment thereof comprising in its variable region at least onecomplementarity determining region (CDR) selected from the groupconsisting of: the heavy chain CDR1 of SEQ ID NO 17, SEQ ID NO 20, SEQID NO: 26, or SEQ ID NO: 32, the heavy chain CDR2 of SEQ ID NO: 18, SEQID NO: 21, SEQ ID NO: 27 or SEQ ID NO: 33, and the heavy chain CDR3 ofSEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 28 or SEQ ID NO: 34, the lightchain CDR1 of SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO:46, or SEQ ID NO: 49, the light chain CDR2 of SEQ ID NO: 24, SEQ ID NO:30, SEQ ID NO: 36, SEQ ID NO: 47, or SEQ ID NO: 50, and the light chainCDR3 of SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 48, orSEQ ID NO:
 51. 6. The antibody or binding fragment of claim 5 comprisingthe amino acid sequence of a heavy chain variable region (VH) selectedfrom the group consisting of: SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 10;SEQ ID NO: 14; SEQ ID NO: 39; SEQ ID NO: 42; and SEQ ID NO: 43 and/or alight chain variable region (VL) selected from the group consisting of:SEQ ID NO: 8; SEQ ID NO: 12; SEQ ID NO: 16; SEQ ID NO: 41; SEQ ID NO:44; and SEQ ID NO:
 45. 7. A composition comprising the binding moleculeof claim
 1. 8. The composition of claim 7, further comprising anadditional agent useful for treating Alzheimer's disease, selected fromthe group consisting of small organic molecules, anti-Abeta antibodies,or any combination thereof.
 9. A kit for the diagnosis of a disorderwhich is characterized by abnormal accumulation and/or deposition of adisorder-associated protein in the brain comprising the binding,molecule of claim 1, the composition of claim 7, or any combinationthereof.
 10. A method for in vivo detection of or targeting atherapeutic and/or diagnostic agent to a disorder-associated protein inthe brain, detecting, suppressing formation of or reducing pathologicalprotein aggregates or conformations in a subject, for improvingcognition or slowing or reversing cognitive decline associated withdiseases, or for extra-corporal extraction of pathological compounds ortheir precursors from body fluids comprising administering the bindingmolecule of claim
 1. 11. A recombinantly produced antibody orantigen-binding fragment thereof which specifically binds: the sameneoepitope of a disorder-associated protein as a reference antibodyselected from the group consisting of NI-101.10, NI-101.11, NI-101.12,NI-101.13, NI-101.12F6A, NI-101.13A, and NI-101.13B.
 12. A recombinantlyproduced antibody or antigen-binding fragment thereof, wherein theantibody competitively inhibits a reference antibody selected from thegroup consisting of NI-101.10, NI-101.11, NI-101.12, NI-101.13,NI-101.12F6A, NI-101.13A, and NI-101.13B from binding to the neoepitopeof a disorder-associated protein.
 13. A recombinantly produced antibodyor antigen-binding fragment thereof wherein the antibody comprises anantigen binding domain identical to that of an antibody selected fromthe group consisting of NI-101.10, NI-101.11, NI-101.12, NI-101.13,NI-101.12F6A, NI-101.13A, and NI-101.13B.
 14. The binding molecule ofclaim 1, which is encoded by a cDNA, or fragment, variant, or derivativethereof, wherein the cDNA is derived from B-cells or memory B-cellsobtained from a human patient who is symptom-free but affected with orat risk of developing a disorder, or a human patient with an unusuallystable disease course.